Engineering hiPSCs to enhance neural progenitor transplantation in degenerative cervical myelopathy
Neural progenitor cell transplantation has been widely explored in traumatic spinal cord injury (SCI) but has yet to be evaluated as a treatment for degenerative cervical myelopathy (DCM), a non-traumatic form of spinal cord injury. Importantly, the extent of astrogliosis and extracellular matrix remodelling that could limit or enhance neural progenitor cell (NPC) graft efficacy in DCM is unknown. The first aim of this project was to examine changes in the extracellular matrix in DCM. Immunohistochemistry of post-mortem patient tissue and a mouse model of DCM indicated astrogliosis and chondroitin sulfate proteoglycan accumulation, which could inhibit NPC integration. Meanwhile, bulk-transcriptomics from human post-mortem tissue revealed no upregulation of astrogliosis- or fibrosis-related genes in DCM. Second, the extent of endogenous remodelling of perineuronal extracellular matrix structures that could enable functional connectivity of NPC grafts – and which may underlie compensatory plasticity in DCM – is unknown. Immunohistochemical analysis of perineuronal nets (PNN) in murine models of DCM showed no significant changes in the number of PNN-positive neurons. Additionally, transcriptomic analysis of PNN-related gene expression from human tissue similarly showed no significant changes in DCM. Therefore, while diffuse astrogliosis and ECM deposition occurs in DCM, endogenous PNN remodelling may not underlie compensatory plasticity. Next, given the importance of PNNs in regulating synaptic plasticity, elucidating mechanisms of their development and remodeling through in-vitro models of human neurons would be useful. No such systems have been reported thus far and it remains unclear whether forward reprogrammed hiPSC-derived glutamatergic and GABAergic neurons contain PNNs. The second aim of this thesis was to determine the extent of PNN formation in forward reprogrammed neurons. Both NGN2 (glutamatergic) and ASCL1/DLX2 (GABAergic) lines displayed punctate, immature PNN structures immunopositive for WFA, which increased along with aggrecan expression over 28 days post-induction. Analysis of existing single-cell and bulk-transcriptomic datasets revealed maturity-dependent increases in neurocan expression, a transient increase in brevican, and no significant increases in aggrecan expression, suggesting that the lectican responsible for WFA immunopositivity in these cells is likely neurocan. Finally, inducible expression of GDNF and ChABC from transplanted NPCs in DCM has the potential to improve graft survival and integration, while providing neuroprotective and regenerative benefits for host cells. Therefore, a dual safe-harbour tetracycline-inducible control system was used to express GDNF and ChABC in human induced pluripotent stem cells (hiPSC) and hiPSC-derived neural progenitors. Immunoblotting, enzyme linked immunosorbent assays, and qPCR were used to measure expression of GDNF and ChABC in-vitro, along with functional assays for ChABC. These established dox-inducible expression and secretion of GDNF and functional ChABC at both the iPSC and NPC stages. This system enables robust regulatable expression of GDNF and ChABC in hiPSC-derived NPCs and provides a path towards further evaluation of engineered NPCs in pre-clinical models of DCM and SCI.
Degenerative Cervical Myelopathy, Spinal cord injury
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