Modelling of the Human Corticospinal Motor Axis and Its Injury Using Organoids

Abstract

Spinal cord injury (SCI) is a debilitating disease which lacks viable treatment options. Thus, affected individuals may find themselves both functionally and socioeconomically disadvantaged with limited therapeutic avenues to explore. While there has been great progress in understanding the pathology of SCI, at heart, the human central nervous system (CNS) is both complex and its regenerative capacity restricted. Employed models to study SCI are typically rodent systems. These models differ in neuroanatomy, cell complexity, and inflammatory response. Consequently, while assumptions can be made over conserved mechanisms, our understanding of human-specific response to trauma is lacking. Furthermore, the translatability of therapeutics which have shown promise in treating SCI in rodent models needs validation. Therefore an in vitro model of the human corticospinal neuraxis could serve as a system to investigate both human specific response to injury and further assess previously identified therapeutics from animal studies. In this work, development of such a system is shown, utilising stem cell derived cerebral and spinal organoids to recreate the corticospinal neuraxis and traumatic injury responses. To develop this corticospinal neuraxis, an optimised spinal organoid differentiation protocol was established. Adapting from previous protocols, its shown that by modulating Sonic Hedgehog and growth factor signalling, organoids with different ventral identities can be produced. After finalising a differentiation protocol producing enriched populations of key circuit forming cells, such as V2a interneurons and motorneurons, spinal organoids were further explored for cell composition utilising single cell RNA-sequencing. These organoids contain multiple cell types found in the native spinal cord such as astrocytes, oligodendrocytes, and an array of neurons. Then, utilising a novel co-culture system, connections could be formed between cerebral and spinal organoids recreating the corticospinal neuraxis. These cultures, termed human corticospinal neuraxis models (hCSNM), were evaluated using immunohistochemistry and electrophysiology to show functional connectivity between organoids and the ability to innervate co-cultured muscle. Finally, injury response was explored in isolated spinal organoids before developing an assay in hCSNMs to look at post-injury axonal growth using 2-Photon imaging to screen growth promoting compounds, validating the potential growth promoting effect of PTEN-inhibition.