Human iPSC-Derived Cerebral Organoids for In Vitro Stroke Modelling and Therapeutic Testing

Background Traditional in vivo animal models often fail to accurately replicate the complexity and molecular responses to cerebral ischaemia. Human induced pluripotent stem cell (h-iPSC)-derived cerebral organoids offer a promising alternative to rodents to circumvent the issue with species divergence. The aim of this research is to develop and characterise h-iPSC-derived cerebral organoids as a human in vitro model of ischaemic stroke using chemical induction and oxygen-glucose deprivation (OGD). Methods Cerebral organoids were generated from control h-iPSC lines (gifted by Washington University) using modified Bowles and Bertucci protocols and cultured under standard conditions. This is work in progress with the following end-points: assessment for pluripotency (qPCR – OCT4, SOX2 and NANOG, Immunostaining – SSEA-4, TRA-1-60 and TRA-1-81, and flow cytometry - SSEA-4, TRA-1-60 and TRA-1-81), karyotyping (chromosomal analysis), functional analysis (metabolic activity assay) and organoid size measured at days 3, 6, 14, and 20 to check for linear growth then and days 20, 40, 60, 80, 100, and 120 in vitro. Results and Conclusions Cerebral organoids show changes in diameter (µm) over time as observed on D3, D6 and D9. Although, with only three data points, the linearity isn’t definitive. Growth appears roughly linear increasing by approximately 30 µm every 3 days. At this stage, it can be deduced or concluded that moderate linear growth is observed, which suggests an early to mid-growth phase. Once cerebral organoids have undergone quality control, the organoids will be subjected to excitotoxins or OGD (1% O2 in glucose-free media for 4 hours) with and without candidate therapeutic agents and assessed at 24 hours for neuronal damage using cell viability assays for validation as a reproducible human-based stroke model to advance mechanistic studies and to serve as a preclinical tool for testing candidate therapeutics.