University Medical Center Goettingen Göttingen, Germany
Abstract: Nerve growth factor (NGF) is a neurotrophin crucial for the differentiation and survival of sympathetic neurons (SNs). NGF released by perivascular cells in the developing heart, controls cardiac sympathetic innervation. We developed a 3D innervated cardiac muscle model from hiPSC (iEHM) by fusing a sympathetic neuronal organoid and engineered heart muscle. Using optogenetics, we demonstrated functional connectivity between SN and cardiac cells (n=41 iEHM, 3 independent differentiations). Single nuclei RNA sequencing revealed among others the co-development of vascular and perivascular cells which were further validated by immunofluorescence. The extensive vascular network (PECAM1pos), surrounded by pericytes (PDGFR-betapos) was interlaced by neurons and sympathetic varicosities (SYN1/THpos) suggesting an interaction between SN and vascular cells. Orientation and proximity analysis, via machine learning-based system, showed a significant correlation in the directionality of SN and cardiac vessels in iEHM. snRNAseq showed that perivascular cells in the tissue were the main source of NGF, which is in line with in vivo data. To study the role of NGF in neuro-cardio-vascular development, we developed two hiPSC lines via CRISPR/Cas9 engineering: an NGF knock-out (NGF-KO) and an inducible NGF (iNGF). For iNGF, a TET-ON system linked to an NGF coding sequence and green fluorescence protein was integrated into the AAVS1 locus. Correct clones from both lines were validated by Sanger sequencing and characterized for stemness, karyotyping, germ layer differentiation, and function. NGF-KO was confirmed by qPCR in bioengineered neuronal organoids (BENO) that typically express NGF. For iNGF, green fluorescence following doxycycline treatment was detected in hiPSC and cardiomyocytes, indicating NGF induction. NGF quantification by ELISA validated NGF secretion with supernatant levels at 2944 pg/mL (n=15) vs. control at 10 pg/mL (n=6). Preliminary iEHM experiments showed that NGF induction led to an increase in cardiac muscle innervation compared to control while NGFKO to a reduction, supporting the role of NGF in human cardiac innervation. In the future, we will employ these tools to investigate neuro- cardio-vascular development and cell-to-cell interactions in iEHM.
