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

Abstract

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 two-compartment 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.

Unravelling the signaling power of pollutants

“…Current attempts to improve organoids aim to integrate macrophages and endothelial cells in organoids to study inflammatory and resident cell interactions [77]. Organs-on-chips or multiorgan fluidics are another type of 3D modeling system aiming to culture basic functional units on a micrometer-sized chamber [76,78,79]. In contrast to other models, microfluidic chip devices can be designed to recreate interactions between two or more different cell types.…”

Portrait of intense communications within microfluidic neural networks

“…Since the first demonstrations, PDMS-based microfluidics has been used for modeling brain circuits on a chip 14 – 17 , as well as for single-neuron analysis 18 – 22 . This approach combines neuron-adhesive coating and physical barriers for efficient cell adhesion and time-stable architectures 13 , 23 – 26 while maintaining high optical transparency for high-resolution imaging 27 , 28 . Additionally, microfluidic devices can be assembled with any substrate, including electrical device arrays 4 , 29 – 34 for monitoring the activity of the same cell and how its activity evolves over time.…”

Functional engineering of human iPSC‐derived parasympathetic neurons enhances responsiveness to gastrointestinal hormones

“…Human‐derived cells inherently reflect our unique genetic and metabolic intricacies, ensuring a more accurate representation of human biology in experimental outcomes, potentially leading to more relevant therapeutic discoveries. Recent efforts have focused on utilizing in vitro approaches to generate human sensory [ 12 , 13 , 14 , 15 ], enteric [ 16 , 17 , 18 ], and autonomic nerves [ 19 , 20 , 21 , 22 , 23 ] from human embryonic stem cells (ESCs) and iPSCs. However, a clear limitation in these efforts is the absence of nerves responsive to GI hormones, which limits our ability to explore the intricate brain–gut axis and understand the etiology and potential interventions for metabolic or neurodegenerative diseases.…”