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

Vascular cell development from the human epicardium is regulated by transient expression of SHISA3 during early transition

L. Priesmeier1, D. Hartung1, E. Schoger1, F. Bleckwedel2, L. Cyganek3, L. Zelarayán1, für die Studiengruppe: AG31

1Institut für Pharmakologie und Toxikologie, Universitätsmedizin Göttingen, Göttingen; 2Pharmakologie und Toxikologie, Universitätsmedizin Göttingen, Göttingen; 3Herzzentrum Göttingen - Stem Cell Unit, Universitätsmedizin Göttingen, Göttingen;

Unraveling the developmental mechanisms of vascular cells can be utilized to define novel sources of endogenous cell renewal in the diseased heart, whereas the epicardium has emerged as an interesting structure to target in cardiac regeneration. We have previously demonstrated that Wnt/β-catenin activation resulted in cardiomyocyte (CM) and vascular cell fetal reprogramming leading to pathological remodeling in the adult heart. In this context, we revealed the presence of a so far undiscovered non-CM SHISA3 expressing cell population in the developing and diseased heart found in the subepcardium of fetal hearts and in the interstitium of pressure overloaded and ischemic hearts. We hypothesize that SHISA3+ cells are a transiently existing population of subepicardial progenitors and aimed to characterize them in human vascular development. In the developing mouse heart, SHISA3+ cells co-expressing alpha smooth muscle actin were identified primarily in the subepicardium at embryonic day (E)14.5, co-localizing with immature vascular cell markers. Resembling our in vivo findings, human induced pluripotent stem cells (hiPSCs) differentiated towards epicardial cells and their derivatives, including vascular smooth muscle cells (vSMCs) and fibroblasts, showed early upregulation of SHISA3 followed by a progressive loss. To further validate the SHISA3+ population as a putative common vascular progenitor cell pool, whole cell single cell sequencing was performed at differentiation day 8, revealing as a marker gene expressed exclusively in cells in early transition, evolving from an epicardial identity (CDH1, SEMA3D, ERBB3) towards a mesenchymal cell fate (FN1, SMOC2, TCF21). In order to decipher the effects of SHISA3 overexpression in a timely controlled manner in our developmental models, we generated and fully characterized transgenic hiPSC lines for CRISPR activation-based doxycycline (dox) inducible endogenous SHISA3 activation, along with a non-targeting guide RNA cell line as control. Differentiation towards epicardial-derived vSMCs upon dox-induced upregulation of SHISA3 expression followed by whole transcriptome RNA analysis revealed regulation of TGF-beta as well as p53 signaling pathways. As these pathways constitute integral components of the epithelial to mesenchymal transition (EMT) process, this characterizes SHISA3 as a putative regulator of vascular cell development from the epicardium. Moreover, epicardial cells treated with SB-431542, a potent inhibitor of TGF-beta signaling, showed maintained expression of SHISA3 throughout development, indicating a mutual control mechanism. Our results showed that reactivation of SHISA3-expressing cells as a novel cardiovascular progenitor pool may be part of the compensatory fetal reprogramming upon pathological heart remodeling. We were able to identify SHISA3 as a marker of early progenitor cells in transition, deriving from the epicardium towards a vascular cell fate. The upregulation of endogenous SHISA3 expression in early vSMC development from our novel hiPSC line revealed a SHISA3-dependent regulation of essential EMT inducing pathways. This strongly indicates its contribution to cell fate determination during epicardial EMT driven vascular cell development. Currently, hiPSC-derived epicardial cells with a SHISA3 loss-of-function are examined, enabling us to further decipher the processes of human cardiogenesis and their reactivation in cardiac disease.

https://dgk.org/kongress_programme/ht2023/aBS129.html