Pluripotent stem cell derived airway cholinergic neurons for disease modelling of neuroplasticity in asthma

Abstract

Rationale: Neuroplasticity plays an essential role in asthma. To work towards a novel and relevant human model, our aims are (1) to establish a protocol for human pluripotent stem cell (hPSC) derived airway cholinergic neurons and (2) to develop a microfluidic lung-on-a-chip which would mimic neuro-effector interactions of the airways.

Results: hPSCs were differentiated towards vagal neural crest (NC) precursors using dual SMAD inhibition and Wnt activation. These precursors were further differentiated towards airway cholinergic nerves using the neurotrophin BDNF. FACS analysis showed that >95% of the hPSCs converted to a NC precursor (HNK1-p75++). The presence of vagal NC precursors was confirmed by HOXB3 and HOXB5 expression. Differentiation of the vagal NC towards airway nerves resulted in 15-45% β3-Tub-ChAT++ population, demonstrating a specific cholinergic neuronal phenotype. Neurons showed electrical activity on a multi-electrode array and responded to ACh and KCl with changes in Ca2+ mobilization

A PDMS microfluidic chip was fabricated with two main compartments, to separate cell bodies of the neurons from the ASM cells. The two compartments were connected via microchannels, to enable neuro-effector communication between the two cell types via axons. When the hPSC-derived cholinergic neurons were cultured in the chip, the axonal outgrowth was visible, while the somatic bodies of the neurons where confined to their own compartment, allowing the study of neuro-effector interactions.

Conclusions: We successfully developed a differentiation protocol for human airway cholinergic neurons from hPSCs and a two-compartment chip for the study of airway neuroplasticity in asthma.