FUNCTIONAL CHARACTERIZATION AND ORGAN-ON-CHIP INTEGRATION OF HUMAN IPSC-DERIVED SYMPATHETIC NEURONS

The peripheral nervous system (PNS) influences the function of nearly all tissues by regulating various physiological processes. When compared to the central nervous system (CNS) neurons, an equivalent understanding of the functional properties of human PNS neurons is lacking in vitro. Generating subtypes of human PNS neurons opens possibilities for Organ-on-Chip (OoC) models with innervation to study both natural organ development and function in disease stages.
First, we modified an existing differentiation protocol for human induced pluripotent stem cell (hiPSC)-derived sympathetic neurons to enable their use in OoC models. Expression of specific neuronal markers was studied with immunocytochemistry, gene expression with qPCR and RNA-seq., and neurotransmitter secretion with ELISA. Functional characterization was performed with multielectrode array (MEA) measurements combined with pharmacological treatments in combination with RNA seq, and synapse quantification. Moreover, the innervation capacity of sympathetic neurons was evaluated in multiple OoC platforms.
Sympathetic neurons expressed cell type-specific markers at gene and protein levels. Synaptic structures formed alongside cholinergic acetylcholine receptors, consistent with sympathetic neuron identity. Neurons exhibited spontaneous activity on MEAs, with distinct electrophysiological and genetic features compared with hiPSC-derived cortical neurons. Neurons also responded to excitatory and inhibitory pharmacology specific to sympathetic neurons, and secreted appropriate neurotransmitters. Neurons were successfully integrated into OoC’s with human cardiomyocytes and differentiating adipose-derived stem cells, forming functional innervation that enhanced target cell maturation. These results provide knowledge on functional properties of human sympathetic neurons and support the establishment of innervated multiculture OoC models for health and disease.