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. In
asthma and other diseases of the respiratory tract, airway cholinergic neurons
undergo plasticity and contribute to airway hyperresponsiveness and mucus
secretion. We currently lack human in vitro models for airway cholinergic
neurons. Here, we aimed to develop a human in vitro model for peripheral
cholinergic neurons using human pluripotent stem cell (hPSC) technology.
hPSCs were differentiated towards vagal neural crest precursors and
subsequently directed towards functional airway cholinergic neurons using
the neurotrophin brain-derived neurotrophic factor (BDNF). Cholinergic
neurons were characterized by ChAT and VAChT expression, and responded
to chemical stimulation with changes in Ca2+ mobilization. To culture these
cells, allowing axonal separation from the neuronal cell bodies, a twocompartment PDMS microfluidic chip was subsequently fabricated. The two
compartments were connected via microchannels to enable axonal outgrowth.
On-chip cell culture did not compromise phenotypical characteristics of the
cells compared to standard culture plates. When the hPSC-derived peripheral
cholinergic 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 the
axonal compartment. The microfluidic chip developed in this study represents a
human in vitro platform to model neuro-effector interactions in the airways that
may be used for mechanistic studies into neuroplasticity in asthma and other
lung diseases.