A human in vitro model for airway neuro-effector interactions

Airway nerves play a key role both in the development of the bronchial hyper-responsiveness and in chronic cough associated with asthma. However, there is an inherent difficulty in studying complex cell-cell interactions that characterize neuro-effector responses in asthma using laboratory animals. Due to recent technological advances, it is now possible to develop a human in vitro model by combining human pluripotent stem cell (hPSC) and organ-on-a-chip technology. The results of this thesis show that we are able to develop a protocol in which we differentiated hPS cells into cholinergic airway nerves. First, the hPS cells were differentiated to a vagal neural crest precursor, and then to cholinergic airway nerves using the neurotrophin BDNF. About 30% of the hPS cells were converted into these airway nerve cells, which is quite efficient. This was confirmed by expression of β-3-tubulin and ChAT, special markers for cholinergic neurons. Furthermore, neurons responded to acetylcholine and potassium chloride (KCl), demonstrating that they are also functional. Subsequently, a PDMS microfluidic chip was fabricated to further study nervous system function. Airway smooth muscle cells play an important role in bronchial hyperresponsiveness. Neurons and smooth muscle cells were cultured in the individual compartments in the chip, which were connected via microchannels to allow axonal communication, as happens in the body. When cholinergic neurons and smooth muscle cells were cultured in the chip, the axons innervated the smooth muscle cells in the other compartment, enabling the study of neuronal communication. In conclusion, this research shows that it is possible to make a human model to enable communication between the nervous system and the lungs on a chip

Human pluripotent stem cells for the modelling and treatment of respiratory diseases

Respiratory diseases are among the leading causes of morbidity and mortality worldwide, representing a major unmet medical need. New chemical entities rarely make it into the clinic to treat respiratory diseases, which is partially due to a lack of adequate predictive disease models and the limited availability of human lung tissues to model respiratory disease. Human pluripotent stem cells (hPSCs) may help fill this gap by serving as a scalable human in vitro model. In addition, human in vitro models of rare genetic mutations can be generated using hPSCs. hPSC-derived epithelial cells and organoids have already shown great potential for the understanding of disease mechanisms, for finding new potential targets by using high-throughput screening platforms, and for personalised treatments. These potentials can also be applied to other hPSC-derived lung cell types in the future. In this review, we will discuss how hPSCs have brought, and may continue to bring, major changes to the field of respiratory diseases by understanding the molecular mechanisms of the pathology and by finding efficient therapeutics

Advanced Modeling of Peripheral Neuro-Effector Communication and -Plasticity

The peripheral nervous system (PNS) plays crucial roles in physiology and disease. Neuro-effector communication and neuroplasticity of the PNS are poorly studied, since suitable models are lacking. The emergence of human pluripotent stem cells (hPSCs) has great promise to resolve this deficit. hPSC-derived PNS neurons, integrated into organ-on-a-chip systems or organoid cultures, allow co-cultures with cells of the local microenvironment to study neuro-effector interactions and to probe mechanisms underlying neuroplasticity

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