Microfluidic and optogenetic technologies to model spinal cord development and neuromuscular junction formation and function

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2015."June 2015." Cataloged from PDF version of thesis.Includes bibliographical references (pages 106-118).Motor neurons located in the spinal cord and innervating muscle cells throughout the body are responsible for virtually all motor functions, from locomotion to respiration or speech. They arise from differentiation of progenitor cells within the neural tube under spatiotemporally well-defined morphogen concentration profiles, and extend axons into the peripheral nervous system following a precisely orchestrated sequence of events involving secreted chemo-attractants and repellents and dynamic expression of the corresponding ligand receptors. Finally, they form neuromuscular junctions, the synapses that transmit electrical signals to the muscle effectors. Failure for these motor neurons to develop or function properly, caused by developmental or neurodegenerative genetic disorders, or as a result of traumatic injuries, lead to highly incapacitating or even lethal malformation and conditions. Microfabricated platforms and optogenetic technologies have proven to be valuable tools to control the microenvironment, biochemical cues and the stimulation applied to neuronal tissues. Precise control of the geometry of microfluidic devices together with their ability to host 3D cell culture has enhanced the physiological relevance of such neuronal tissues relative to traditional 2D culture assays. And the ability to selectively excite neuronal cells with light has opened tremendous opportunities in the field of neuroscience. In this thesis, we combine these two technologies to stimulate and subject cells to chemical and physical microenvironments that emulate their in vivo counterpart. First, we present a microfluidic platform that generates orthogonal concentration gradients and emulates the confined appearance of motor neurons within the developing spinal cord. Then, we introduce a new device capable of forming a 3D compartmentalized neuron-muscle coculture and demonstrate remote stimulation of the myofibers by the motor neurons resulting in muscle contraction. By targeting the stem cells from which the motor neurons are derived with the light sensitive ion channel Channelrhodopsin, we form, in this microfluidic device, the first in vitro light-activatable neuromuscular junction. Keywords: microfluidics, optogenetics, morphogenesis, cell migration, neuromuscular junctions.by Sébastien G. M. Uzel.Ph. D