Automated microfluidic cell culture of stem cell derived dopaminergic neurons.

Parkinson's disease is a slowly progressive neurodegenerative disease characterised by dysfunction and death of selectively vulnerable midbrain dopaminergic neurons and the development of human in vitro cellular models of the disease is a major challenge in Parkinson's disease research. We constructed an automated cell culture platform optimised for long-term maintenance and monitoring of different cells in three dimensional microfluidic cell culture devices. The system can be flexibly adapted to various experimental protocols and features time-lapse imaging microscopy for quality control and electrophysiology monitoring to assess cellular activity. Using this system, we continuously monitored the differentiation of Parkinson's disease patient derived human neuroepithelial stem cells into midbrain specific dopaminergic neurons. Calcium imaging confirmed the electrophysiological activity of differentiated neurons and immunostaining confirmed the efficiency of the differentiation protocol. This system is the first example of an automated Organ-on-a-Chip culture and has the potential to enable a versatile array of in vitro experiments for patient-specific disease modelling.

Derivation of Human Midbrain-Specific Organoids from Neuroepithelial Stem Cells.

Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson's disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.

Sonographic Dynamic Description of the Laryngeal Tract: Definition of Quantitative Measures to Characterize Vocal Fold Motion and Estimation of Their Normal Values.

Vocal fold motion was analyzed during free breathing using two-dimensional dynamic ultrasound imaging. Two cadavers were first analyzed to define easily identifiable landmarks. Motion of the laryngeal tract was then analyzed in an axial plane. Left and right arytenoids and thyroid cartilage were defined on images corresponding to abduction and adduction of the laryngeal tract. Associated area measurements were established for 50 healthy subjects. All area indices were significantly larger during abduction than adduction. Symmetry of motion was established by comparing each hemi-larynx, and mobility fractions were defined. Normal values of laryngeal motion during free breathing were thus established.

A constraint-based modelling approach to metabolic dysfunction in Parkinson's disease.

One of the hallmarks of sporadic Parkinson's disease is degeneration of dopaminergic neurons in the pars compacta of the substantia nigra. The aetiopathogenesis of this degeneration is still not fully understood, with dysfunction of many biochemical pathways in different subsystems suggested to be involved. Recent advances in constraint-based modelling approaches hold great potential to systematically examine the relative contribution of dysfunction in disparate pathways to dopaminergic neuronal degeneration, but few studies have employed these methods in Parkinson's disease research. Therefore, this review outlines a framework for future constraint-based modelling of dopaminergic neuronal metabolism to decipher the multi-factorial mechanisms underlying the neuronal pathology of Parkinson's disease.

Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.

A hallmark of Parkinson's disease is the progressive loss of nigrostriatal dopaminergic neurons. We derived human neuroepithelial cells from induced pluripotent stem cells and successfully differentiated them into dopaminergic neurons within phase-guided, three-dimensional microfluidic cell culture bioreactors. After 30 days of differentiation within the microfluidic bioreactors, in situ morphological, immunocytochemical and calcium imaging confirmed the presence of dopaminergic neurons that were spontaneously electrophysiologically active, a characteristic feature of nigrostriatal dopaminergic neurons in vivo. Differentiation was as efficient as in macroscopic culture, with up to 19% of differentiated neurons immunoreactive for tyrosine hydroxylase, the penultimate enzyme in the synthesis of dopamine. This new microfluidic cell culture model integrates the latest innovations in developmental biology and microfluidic cell culture to generate a biologically realistic and economically efficient route to personalised drug discovery for Parkinson's disease.