Abstract 295 Generation of a doxycyline inducible CRISPR-based gene activator human induced pluripotent stem cell line to characterize SHISA3, a novel Wnt-dependent signature in the developing cardiac vasculature

Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02087-y
Generation of a doxycyline inducible CRISPR-based gene activator human induced pluripotent stem cell line to characterize SHISA3, a novel Wnt-dependent signature in the developing cardiac vasculature
L. Priesmeier¹, D. Hartung¹, E. Schoger¹, F. Raad¹, D. Panakova², L. Cyganek³, L. Zelarayán¹
¹Institut für Pharmakologie und Toxikologie, Universitätsmedizin Göttingen, Göttingen; ²Max-Delbrück-Centrum für Molekulare Medizin, Berlin; ³Herzzentrum Göttingen - Stem Cell Unit, Universitätsmedizin Göttingen, Göttingen;
Understanding the developmental processes of vascular cells will aid to dissect mechanisms of neovascularization in the failing heart. We have previously demonstrated that Wnt/β-catenin activation resulted in cardiomyocyte and vascular cell fetal reprogramming leading to pathological remodeling in the adult heart. This was accompanied by an increased SHISA3 expressing cell population, a novel developmental cell type in heart failure. Using a SHISA3 overexpression human induced pluripotent stem cell (hiPSC) line and various human in vitro studies, we now aimed at identifying the role of these cells in human vascular cell fate. Modulating Wnt signaling in transgenic mouse models revealed a so far uncharacterized non-cardiomyocyte (non-CM) cell type expressing SHISA3, which was increased upon Wnt activation. These cells were found highly expressed in the developing mouse and hypertrophic adult mouse and human hearts. Immunofluorescence analysis showed SHISA3 expression in non-CM cells at embryonic day (E)9.5-11.5. At fetal E14.5, SHISA3pos cells were located sub-epicardially and later at E18.5, they were abundantly expressed interstitially and in capillary structures in the myocardium. SHISA3pos cells co-expressed actin and aortic smooth muscle (ACTA2) in capillary structures. Progressive co-localization of these cells with the early endothelial marker Endomucin (EMCN) was detected from E10.5-E18.5, which was gradually lost postnatally and reappeared upon pathological remodeling. In human developmental models, we observed that SHISA3 was transiently expressed during endothelial cell differentiation and in a 3D model of cardiovascular development from hiPSCs. Transcriptional time course studies in hiPSC-derived epicardial cells showed early upregulation of SHISA3 along with a progressive loss during further development into various epicardial-derived cell types including vascular smooth muscle cells, endothelial cells and stroma cells, resembling previous in vivo findings in the mouse. This characterized the SHISA3pos population as a putative common vascular progenitor cell pool. To further investigate the effects of enhanced SHISA3 expression on vascular cell programming, we integrated a doxycycline (DOX)-inducible SHISA3 guide (g)- as well as a control non-targeting-gRNA construct along with an EGFP expressing reporter cassette into the ROSA26 locus of a hiPSC line endogenously expressing dead (d)Cas9-VPR-tdTomato for gene activation. GuideRNA efficiency and functionality of our DOX-induction system were validated in HEK293T cells beforehand. We thereby established double transgenic cell lines allowing for inducible SHISA3 upregulation based on enzymatically inactive dCas9 mediated DOX-dependent SHISA3 gRNA expression in a temporally controlled and titratable manner. The hiPSC lines were fully characterized and differentiated towards epicardial cells and their progenies upon DOX-induced upregulation of SHISA3 expression at specific time points of development. Our results showed that reactivation of SHISA3-expressing cells may be part of the compensatory fetal reprogramming upon pathological heart remodeling. This study will test the consequences of time-dependent SHISA3 regulation at different developmental stages of hiPSC-derived epicardium and its vascular progeny. The generated hiPSC lines are used to follow up on SHISA3pos cells in 2D co-culture and 3D models including cardiomyocytes, stroma cells and SHISA3pos epicardial progenitors.
https://dgk.org/kongress_programme/ht2022/aBS657.html