Endogenous gene activation of cell cycle regulators promotes cell cycle re-entry in human induced pluripotent stem cell-derived cardiomyocytes

While adult cardiomyocytes have a limited capacity for proliferation, understanding and manipulating the underlying mechanisms that govern cardiomyocyte cell cycle re-entry, controlled proliferation and re-differentiation could pave the way for innovative regenerative therapies to treat cardiovascular diseases. In this study, we aim to  advance this knowledge by understanding endogenous and context-dependent mechanisms, which are crucial for developing effective, controllable strategies to stimulate cardiomyocyte proliferation. We harnessed the power of the combination of CRISPR/dCas9VPR mediated endogenous gene activation (CRISPRa) and the Fluorescence Ubiquitination-dependent Cell Cycle Indicator (FUCCI) for precise cell cycle activity monitoring. CRISPRa uses an enzymatically inactive Cas9 with transcriptional activators targeted to gene regulatory regions via guide RNA (gRNA) offering the possibility to enhance transcription of multiple genes at once. The FUCCI system includes fluorescently tagged cell cycle regulatory proteins specific to different cell cycle phases (G1 phase: mCherry-tagged Chromatin licensing and DNA replication factor 1 (CDT1)/ G2 phase: ECFP-tagged Geminin). The oscillation of CDT1 and Geminin throughout the cell cycle allows for identification of bona fide cell cycle progression. The FUCCI system was integrated by CRISPR/Cas9 homology-directed repair into the safe harbor ROSA26 locus of endogenously citrine-tagged Alpha- actinin-2 (ACTN2) reporter hiPSC line (Myocyte Cell Cycle Reporter = Myo-CCER). Cell cycle activity monitoring of hiPSC-derived cardiomyocytes (hiPSC-CM) using FUCCI was validated using Wnt signaling activator CHIR99021 (3 µM) for 6 days in 1-week-old hiPSC-CMs. This resulted in cell cycle re-entry as shown by a 5.13-fold increase in ECFP+ nuclei ratio in ACTN2-expressing cells compared to control. To examine the effects of cyclins, CRISPRa was used to endogenously activate cyclins ( CCNA2 and CCNB1 ). Validated gRNAs were lentivirally delivered resulting in significantly increased expression of cyclins 10 days after transduction compared to control non-targeted (NT) gRNAs. Interestingly, the response was different depending on the stages of hiPSC-CMs (n ≥ 3, CCNA2 in 1-month CRISPRa CM: 5.1-fold to NT in 6-months CRISPRa CM: 15.8-fold to NT). The immunofluorescence results indicated that elevated expression of CCNA2 and CCNB1 induced repetitive karyokinesis (> 10 nuclei in a single CM) along with increased G2 phase hiPSC-CM’s. To combine the CRISPR activation system with cardiomyocyte-specific cell cycle activity tracking, the CRISPRa system was integrated into the AAVS1 locus of Myo-CCER resulting in a triple transgene hiPSC line (CRISPRa Cell Cycle Reporter = CraCCER). This cell line is currently used for hiPSC-CM differentiation and 3D engineered human myocardium (EHM) generation to evaluate cell cycle induction in a setting of more mature FUCCI CM. To compare these findings in an in vivo context, we generated an all-in-one AAV9 construct containing FUCCI reporter with Ccna2 CRISPRa gRNAs which will be used for further investigation of the  role of Ccna2 in vivo . In conclusion, we demonstrated cell cycle re-entry of hiPSC-CMs  via independent endogenous CCNA2 and CCNB1 activation. Furthermore, the combined FUCCI/CRISPRa tool is a valuable asset to the field of heart regeneration and can be further employed to decipher mechanisms of cardiomyocyte proliferation in an unbiased approach.