Developing a human cerebral organoid ischaemia model to investigate the response of oligodendrocytes to ischaemia

Stroke is the second leading cause of disability and the third leading cause of death globally. In Norway, there are 11 000 new cases of stroke each year, and 80-85 % of these cases are caused by cerebral artery blood clots or emboli and are termed ischaemic. The current treatment options for ischaemic stroke are limited, and there is a need for new treatments that can reduce brain tissue damage after a stroke. Unfortunately, several neuroprotective agents shown to rescue the stroke-affected tissue in mice have had little to no effect when tested in humans. This suggests differences in stroke pathophysiology between humans and mice, urging the scientific community to produce new experimental models to bridge this gap. Ischaemic stroke leads to cell death distal to the affected blood vessel, including oligodendrocytes. The main job of oligodendrocytes is to myelinate the neuronal axons, working as electrical insulation. Previous studies suggest that oligodendrocytes are particularly vulnerable to ischaemia. Thus, saving oligodendrocytes may be a significant factor in protecting brain tissue after an ischaemic stroke. The major aim of this thesis was to develop a human cerebral organoid ischaemia model to study the response of oligodendrocytes to ischaemia. To achieve this, I aimed to find the optimal ischaemia duration to achieve (1) a significant increase in dead cells after ischaemia compared with control, (2) a significant reduction in oligodendrocytes after ischaemia compared with control, and (3) a significant change in the number of proliferating oligodendrocytes after ischaemia compared with control. Human cerebral organoids were produced from human induced pluripotent stem cells and were used as a model for the human brain. In order to develop an ischaemia model, we tested different durations of incubation in glucose- and oxygen-free conditions. By TUNEL assay, I show that 1, 2 or 3 hours of ischaemia (i.e. oxygen- and glucose deprivation) is insufficient to cause a significant increase in cell death in the organoids. Only at 24 hours of ischaemia did we find significantly more cell death compared with control; however, more time points between 3 and 24 hours should be tested. I used immunohistochemistry to test the effect of ischaemia on oligodendrocyte lineage cells and to look at proliferation 3 days after ischaemia. My results show that there is a large variation between organoids in the number of oligodendrocyte lineage cells that are present. There was no significant difference in the number of oligodendrocytes after 2 or 24 hours of ischaemia compared with control. I found that there was a 45 % and 56 % reduction in the number of proliferating cells after 2 and 24 hours of ischaemia, respectively, compared with control. However, there was no significant difference in the number of proliferating oligodendrocytes between 2 or 24 hours of ischaemia and control