Training in hIPSC differentiation protocols to generate motor neuron-muscle cultures to replace rat models in study of mitochondria on axon physiology

Neurons are the main computational units in the brain. They form a network of interconnected cells that pass information to each other at specialised sites called synapses. Electrical signals known as action potentials, the main currency of information in the neuron, travel down cable-like thin structure called the axon to reach the synapse. Here, they cause the release of chemicals that then result in electrical signals in the receiving neuron. The study of the spreading of electrical signals down axons has been of interest to many researchers over the past 100 years, and Hodgkin and Huxley won the Nobel Prize in 1963 for their identification of the mechanisms that underlie the propagation of these electrical signals. Though their model has been the cornerstone of electrical signal propagation ever since, the equations rely viewing the axon on a relatively large, macroscopic scale. Here we provide preliminary evidence that when viewed on a more microscopic scale, that previously unidentified factors have important influences on how electrical signals are spread down the axon.

Mitochondria are structures that reside within cells to produce energy and soak up excess calcium. Mitochondria occur at regular points along an axon, and due to the relative thin diameter of axons mitochondria almost fill the entire cable when looked at a cross section. According to the Hodgkin-Huxely model this would increase the resistance in the spread of electrical signals, much like a pipe with a blockage reduces the flow of water. However, by using the very latest techniques in microscopy that allow us to track the spread of voltage as a change in intensity of light, we observed that mitochondria increase the transmission of electrical signals along the axon. This surprising discovery seems to result from the interaction between the electrical voltage generated by mitochondria and the electrical voltage of the axon.

The ability to expand on these results is relatively limited in our current model. The axons of rat neurons that are dissected from the hippocampus region of the brain form complex patterns when they are cultured, weaving in random directions and overlapping with each other. In this proposal Dr Rigby wishes to be trained in the culturing of human induced pluripotent stem cell (hIPSC) derived motor neurons, which have very straight axons that infrequently branch and allow for much simpler acquisition and analysis of the spread of electrical and calcium signals.

hIPSCs are the result of some extraordinary discoveries that skin cells from humans can be turned back into a stem cell-like state, from which they can then be turned into any cell type. Motor neurons are the cells which reside in the spine and cause the contraction and relaxation of muscle. Over the past 10 years the protocols to transform hIPSCs into motor neurons and muscle have been refined so that now researchers, including Dr Rigby's collaborator Dr Ivo Lieberam, can reliably produce human neuromuscular cultures without the need of using embryos or animals. It is these protocols that Dr Rigby wishes to learn.

The use of hIPSCs confer many advantages. Firstly, Dr Rigby has the opportunity to completely cease animal usage in his work and thus address the 'Replacement' aspect of the 3Rs. In addition, by growing the neurons and muscle on microfluidic devices he will be able to make a customizable and transferable model of the human neuromuscular system, that can be of benefit to other researchers, and thereby further reduce the use of animals in neuroscience research. hIPSCs can also be sourced from human patients with particular neurodegenerative disorders. In our proposal we also plan to benefit from this versatility of the hIPSC model by comparing the spread of voltage along axons in hIPSC-derived motor neurons from healthy patients with neurons derived from patients with Amyotrophic Lateral Sclerosis (ALS), the most common form of motor neuron disease

With this fellowship, Dr Rigby aims to become trained in the differentiation of motor neurons and muscle from human induced pluripotent stem cell (hIPSC) lines so that he is able to:
1) Cease the use of animals in his research.
2) Expand on some exciting, preliminary, data suggesting mitochondria influence the propagation of voltage along neuronal axons.
3) Help develop an all human-derived neuromuscular culture on microfluidic systems that can be easily transferred between microscopes. Use the latest forms of optical stimulation and functional measurement to enhance applicability to other researchers' work, thereby further reducing animal use.

Under the expert tutelage of Dr Leiberam at the specialised Centre for Stem Cells & Regenerative Medicine, Dr Rigby will learn how to differentiate motor neurons and muscle from hIPSC lines. Both cell types will be cultured within segregated compartments on microfluidic devices that allow the straight and parallel growth of motor neuron axons along small channels where they will form neuromuscular junctions. Dr Rigby will benefit from these optimised models of axon physiology to study how whole-cell current-clamp induced subthreshold voltage changes, and extracellular- or light-evoked action potentials, propagate along motor neuron axons. Either voltage propagation or calcium dynamics will be studied using the latest genetically encoded indicators, and captured with the ultrafast frame rates achieved by sCMOS cameras. By combining these optical measures with a fluorescent reporter of mitochondria, Dr Rigby will be able to test his preliminary data that suggest mitochondria alter their local cytoplasmic charge to enhance voltage propagation. This model offers significant advantages over the current rat hippocampal model, not least that motor neurons can be derived from patients with neurodegenerative disorders such as ALS, allowing Dr Rigby to find novel perspectives into how healthy and diseased axons differ.

This fellowship will enable Dr Rigby (MR) to become proficiently trained in the differentiation of human induced pluripotent stem cells (hIPSC) into motor neurons so that he can completely replace the use of Wistar rats in his work on functional axonal imaging. In addition, by learning how to differentiate hIPSCs into skeletal myotubes, MR will further eliminate the normally required need for chick or rodent tissue for sources of muscle. MR will personally benefit from these syetms, which will not only provide economic advantages by reducing animal costs, but will confer many technical advanatages over the model currently used, including more precise control over neuronal sub-compartments, and axons with much straighter morphologies that will simplify the acquisition and analysis of functional imaging. Such advantages will provide an optimal model for MR to expand on preliminary experiments that suggest mitochondria influence the propagation of voltage along axons.

By co-culturing neurons and muscle on microfludidic platforms to generate neuromuscular 'biochip' devices, it is hoped that this proposal will help other researchers developing similar models. Whilst a number of groups have started to develop this particular neuromuscular microfluidic technology, none have attempted to fully replace the use of animals. This technology is still quite novel and now is an ideal time to demonstrate, to these groups in particular, the dispensability of animals in such models, whilst still attaining the same goals. This will aid such labs by not only saving them money on animal maintenance costs, but also will provide more human relevance to their work. In order to help other researchers transition to the all-hIPSC neuromuscular microfluidic devices, MR aims to make his work available both in a scientific peer reviewed journal, and also in the pre-publication archive, bioRxiv. In addition, he will act in a 3Rs ambassadorial capacity to present the work nationally and internationally at conferences prior to publication, and will advertise them on the Centre for Developmental Neuroscience website as a resource.

The development of hIPSC-derived neuromuscular microfluidic devices also has the potential to aid researchers other than those developing neuromuscular microfluidic devices. By utilizing his experience of what scientists need when studying axon function, MR plans to incorporate optical methods of activation and functional recording of motor neurons. This would transform the microfluidic devices into an optically controlled functional assay, that could be used as a 'plug-and-play' type system potentially attractive to a variety of research groups studying the physiology of motor neuron axons, or axons in general. A further advantage of the hIPSC model that will be useful for other researchers, is their ability to be derived from patients with neurodegenerative disorders. For example, motor neurons have already been derived from hIPSCs taken from patients with Amyotrophic Lateral Sclerosis (ALS). Thus, by simply changing the source of hIPSCs, these devices could be equally helpful for researchers interested in understating pathological mechanisms of neurodegeneration or scientists requiring a model to screen potential therapies for motor neuron diseases.

By sharing his development of neuromuscular microfluidic devices, and thereby helping other researchers find more approprioate, human relevant, models of axon function, this fellowship will have the potential to vastly increase the pool of skilled scientists, knowledgeable about the 3Rs.