Generation of posterior motor neurons from human pluripotent stem cells

Motor neurons are essential for the propagation of signals which evoke muscle contractility and allow all bodily movements. An inability of motor neurons to function correctly, due to damage or degeneration, can result in multiple movement-based disorders. Generation of motor neurons from the in vitro differentiation of human pluripotent stem cells (hPSCs) has demonstrated a potential cell-replacement treatment for neurodegenerative diseases. However, existing in vitro hPSC differentiation protocols predominantly produce motor neurons of a hindbrain and cervical identity and fail to produce high yields of more posterior, thoracic and lumbar, identities.
Multiple studies have shown that thoracic and lumbar neural tissue have differing developmental origins to their anterior counterparts. During axis extension, a population of bipotential progenitors, termed Neuromesodermal Progenitors (NMPs), give rise to the neural and mesodermal lineages of the posterior axis. Therefore, we demonstrate that to efficiently generate a thoracic motor neuron identity, in vitro, there is an initial requirement to differentiate hPSCs via a NMP intermediate state. In doing so, we fully characterise the signals important for promoting both a neural and motor neuron identity downstream of NMPs. We show that WNT and FGF signals promote a dorsal neural state whilst the inhibition of TGFβ and BMP pathways enhance neuralisation. Further TGFβ and BMP pathways antagonism alongside the stimulation of Shh signalling are further implicated within dorso-ventral patterning to promote a ventral motor neuron identity. Whilst, continued FGF signals are required to promote a stable thoracic identity. To our knowledge, compared to existing protocols, our characterised protocol provides the highest yields of thoracic motor neurons and is reproducible within multiple hPSC cell lines. Furthermore, the hPSC-derived thoracic identity motor neurons are electrophysiologically functional and demonstrate the ability to integrate within the neural tube of chick embryos. Additionally, we have also compared the functionality of both hPSC-derived motor neurons of a hindbrain/cervical identity and thoracic identity. We find that both motor neuron subtypes display no electrophysiological differences. However, upon grafting into thoracic regions of chick embryo neural tubes it appears that hPSC-derived hindbrain/cervical motor neuron progenitors appear to display a greater degree of ectopic behaviours compared to their thoracic motor neuron progenitor counterparts. This suggests that acquisition of positional identity, prior to engraftment, can effect behaviour of transplanted cells in vivo.