Differentiation and on axon-guidance chip culture of human pluripotent stem cell-derived peripheral cholinergic neurons for airway neurobiology studies

Airway cholinergic nerves play a key role in airway physiology and disease. Inasthma and other diseases of the respiratory tract, airway cholinergic neuronsundergo plasticity and contribute to airway hyperresponsiveness and mucussecretion. We currently lack human in vitro models for airway cholinergicneurons. Here, we aimed to develop a human in vitro model for peripheralcholinergic neurons using human pluripotent stem cell (hPSC) technology.hPSCs were differentiated towards vagal neural crest precursors andsubsequently directed towards functional airway cholinergic neurons usingthe neurotrophin brain-derived neurotrophic factor (BDNF). Cholinergicneurons were characterized by ChAT and VAChT expression, and respondedto chemical stimulation with changes in Ca2+ mobilization. To culture thesecells, allowing axonal separation from the neuronal cell bodies, a twocompartment PDMS microfluidic chip was subsequently fabricated. The twocompartments were connected via microchannels to enable axonal outgrowth.On-chip cell culture did not compromise phenotypical characteristics of thecells compared to standard culture plates. When the hPSC-derived peripheralcholinergic neurons were cultured in the chip, axonal outgrowth was visible,while the somal bodies of the neurons were confined to their compartment.Neurons formed contacts with airway smooth muscle cells cultured in theaxonal compartment. The microfluidic chip developed in this study represents ahuman in vitro platform to model neuro-effector interactions in the airways thatmay be used for mechanistic studies into neuroplasticity in asthma and otherlung diseases