Hands, Anna Hephzibah; (2021) Examining the effect of V3 interneurons and astrocytes on embryonic stem cell-derived motor neuron maturation in vitro. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Motor function is fundamental to human survival and behaviour. Consequently, muscle impairment and paralysis can severely impact quality of life, as in patients with Amyotrophic Lateral Sclerosis (ALS) and spinal cord injury. Restoring muscle control following damage to motor circuits has proven an elusive goal. Strategies to artificially restore function to paralysed muscles are currently being investigated, and most methods rely on stimulation of host nerves. However, this is only effective in conditions where motoneurons and Neuromuscular Junctions (NMJs) remain intact, which is not the case in neurodegenerative disorders such as ALS. A novel strategy to overcome muscle paralysis involves two emerging technologies; the use of stem cell replacement strategies and optogenetics. Embryonic Stem Cell (ESC)-derived cells can be genetically manipulated to express light-sensitive genes such as ChannelRhodopsin-2 (ChR2). When grafted into a peripheral nerve within embryoid bodies (EBs), ESC-derived, ChR2-expressing motoneurons can grow axons that form functional NMJs, enabling optical control of muscle contraction by light stimulation of the graft. Surprisingly, engraftment of purified motoneuron aggregates does not result in the formation of functional NMJs. This suggests that other, non-motoneuronal cells within EBs contribute to the ability of ESC-derived motoneurons to mature and functionally innervate host muscle. In this Thesis, I examine the possibility that spontaneous activity arising from intra-graft microcircuits is necessary for motoneuron maturation. To investigate this possibility, I generated co-cultures in vitro with pure populations of ESC-derived motoneurons, astrocytes and V3 interneurons. Immunocytochemistry was used to assess the morphology and synapse formation of motoneurons when cultured alone or in combination with other cell types. The effect of co-culture on the electrophysiological properties and spontaneous activity of motoneurons was investigated by single cell patch-clamping and calcium imaging. My results show that co-culture of motoneurons with astrocytes promotes motoneuron survival and morphological maturation, accelerates their electrophysiological development, increases the density of motoneuronal cholinergic synapses, and enables the development of glutamatergic, motoneuronal spontaneous activity. Motoneurons in astrocyte-containing co-cultures also display spontaneous calcium activity, though calcium activity emerges a week later than spontaneous activity recorded by patch clamping and has different burst characteristics. In contrast to astrocytes, V3 interneurons alone have little, if any, effect on motoneuron maturation. However, together with astrocytes, V3 interneurons accelerate the emergence of spontaneous, glutamatergic activity, alter the density of glutamatergic and cholinergic synapses onto motoneurons, and lead to more mature patterns of spontaneous glutamatergic activity in the motoneuron co-cultures. These results provide insight into the influence of other cell types on motoneuron maturation and suggest that astrocytes and V3 interneurons could be added to motoneurons to produce a more mature, spontaneously active graft for use in cell replacement strategies to overcome muscle paralysis.

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