TGFβ induces changes indicative of neuroplasticity in a novel model of hPSC-derived sensory neurons.

Neuroplasticity is a pathological feature of asthma, characterized by a lowered firing threshold, increased neuron density and neuron length, resulting in hyperresponsiveness. We recently reported an increased presence of mast cells in the direct vicinity of airway neurons in patients with fatal asthma. Unfortunately, there is no adequate model to study human sensory neuron plasticity. To address this, we have developed an <italic>in vitro</italic> human model of sensory neurons and exposed these to TGFβ, a cytokine produced by mast cells and other lung cells and important for remodeling processes. We developed and validated a 40-day protocol to differentiate hPSCs into mature sensory neurons. Subsequently, we exposed these sensory neurons for 5 days to TGFβ and assessed changes in the neurons. Immunofluorescence, flow cytometry, and RNA analysis confirmed successful generation of β3-tubulin⁺ neurons, with ~35% being sensory neurons (TRPV1⁺/NAV1.8⁺) in both standard culture and our microfluidic axon-guidance model. TGFβ treatments altered network structure, resulting in more branching and longer segments. This was supported by RNAseq and GSEA, which showed that TGFβ exposure upregulates gene sets associated with peripheral neuron development (e.g. <italic>ASCL1</italic>, <italic>POU4F1</italic>, <italic>CRLF1</italic>, <italic>PLP1</italic>, <italic>NTRK1</italic>), protein synthesis (e.g. <italic>RPL</italic>, <italic>RPS</italic>) and immune cell recruitment (e.g. <italic>PLAUR</italic>, <italic>RGS16</italic>, <italic>IL11</italic>, <italic>CCL19</italic>). We present a novel <italic>in vitro</italic> model of hPSC-derived sensory neurons that replicates neuroplasticity, offering a valuable tool to study its pathological mechanisms. Our findings show that TGFβ can induce neuroplasticity-like changes within 5 days of exposure, providing a new route to study the onset of neuroplasticity in asthma.