Nerve growth factor (NGF) is a neurotrophin essential for the differentiation and survival of sympathetic neurons (SNs). In the developing heart, NGF released by perivascular cells orchestrates sympathetic innervation. To model this process in vitro, we established a 3D innervated engineered heart muscle (iEHM) by fusing human iPSC-derived sympathetic neuronal organoid (SNO) with engineered heart muscle (EHM). Optogenetic SYN-f-Chrimson SNO fused to the EHM increased beating rate upon light stimulation, demonstrating functional connectivity between SNs and cardiomyocytes (n=41 iEHMs, 3 independent differentiations).

To investigate the cellular composition of this system, single-nuclei RNA sequencing (snRNA-seq) was performed, revealing co-development of vascular and perivascular cells. These findings were validated by immunofluorescence that showed a dense vascular network (PECAM1⁺), supported by pericytes (PDGFRβ⁺) interlaced with neurons and sympathetic varicosities (TH⁺/SYN1⁺), suggesting close neuro-vascular interactions. Quantitative analysis using machine learning–based orientation and proximity analysis demonstrated a strong directional correlation between SNs and vessels. Furthermore, snRNA-seq identified perivascular cells as the main source of NGF, consistent with in vivo data.

To dissect the specific role of NGF in neuro-cardio-vascular development, we engineered a doxycycline-inducible NGF (iNGF) hiPSC line using CRISPR/Cas9. The TET-ON iNGF system, integrated at the AAVS1 locus, couples a NGF coding sequence with a GFP reporter. iNGF-EHMs were fused with optogenetic SNOs and cultured for 4 weeks under NGF induction of 0, 1, 2, or 3 weeks (n=10-11 iEHMs per condition). Light stimulation over 3 weeks NGF induction significantly increased the beating rate, while contractile performance significantly decreased in 2- and 3-week iNGF tissues. Immunofluorescence further showed hyperinnervation (TH⁺/NF⁺) of cardiomyocytes (CTNT⁺) after 1–2 weeks of NGF induction, which declined in 3-week iNGF tissue. Orientation/proximity analysis across doxycycline-treated versus control tissues (n=7-13 imaging fields) revealed reduced neuro–vascular alignment after prolonged NGF induction, supporting the concept that pericyte-derived NGF attracts neuronal axons.
Together, the iNGF-iEHM represents a robust in vitro human model to dissect NGF-dependent mechanisms of sympathetic innervation and neuro-cardio-vascular development. Our results demonstrate acute NGF induction enhances neuro-cardiac connectivity, while chronic exposure disrupts its contractile performance. These findings highlight that balanced neurotrophic signaling is essential for coordinated cardiac development and establish a platform to investigate NGF-driven mechanisms underlying cardiac dysfunction.