Relevant and reproducible in vitro models of human neural injury and disease are needed to investigate neurodevelopmental pathology and damage response. This is particularly pertinent for pathologies with poor translation from animal models, including hypoxic-ischaemic injury (HI) and Down syndrome (DS). DS is a multi-systemic developmental disorder and the most common genetic form of mental disability. In vivo models of DS are limited by species-specific chromosomal and neurodevelopmental differences. Perinatal HI is the leading cause of neonatal mortality and neurological disability. Although several potential treatments have shown efficacy in animal models of HI, this success has not translated to human clinical trials. This work aims to develop and validate improved in vitro methods to investigate human neurodevelopmental damage and disease, with a focus on HI and DS. To better represent tissue architecture, 3-dimensional (3D) hydrogel cultures of human neural stem cells (hNSCs) were developed and optimised. Injury models and viability readouts of HI, calcium-dependent and oxidative stress-induced cell death were established in 2D and 3D cultures. Significantly, neural gene expression and injury response were altered in 3D cultures compared to 2D, emphasising the importance of culture architecture for in vitro studies. DS- and control-induced pluripotent cell (iPSC) lines were generated, characterised, and differentiated towards neural progenitor cells (NPCs). Fewer NPCs were produced by DS-iPSCs than control-iPSCs, reflecting both a reduced rate of NPC proliferation and increased NPC death. DS-neurons displayed abnormal morphology, reduced Ca 2+ activity, and altered gene expression. Both 2D and 3D cultures of DS-NPCs displayed increased susceptibility to oxidative stress, whereas DS-astrocytes displayed a decreased vulnerability to oxidative stress and Ca 2+-dependent injury. These findings support the hypotheses that DS-NPC proliferative impairment and susceptibility to oxidative stress contribute to DS neurodevelopmental impairments. Further, these results demonstrate a proof-of-principle for a 3D in vitro model system for the investigation of neural cell injury and neurodevelopmental disorder
