Apoptosis and DNA Repair in Irradiated Cerebral Organoids

Abstract

In the treatment of CNS neoplasms, radiation therapy to high biologically effective doses is performed in both fractionated and single fraction schemes. Inevitably, normal CNS tissues are irradiated, leading to both acute and late toxicities, and demanding greater insight into normal tissue effects and development of innovative strategies for radioprotection and mitigation. For this purpose, the cerebral organoid, which recapitulates many cell types and complex architectures of the human brain, is proposed as a model system. The objective of our study was to characterize the response of cerebral organoids to high single fraction doses of photon irradiation in terms of apoptosis, DNA damage, and activation of DNA repair.Cerebral organoids were prepared from human embryonic stem cells as previously described. CNS tissue differentiation was verified by histopathology. Irradiation of organoids was accomplished using a 160 kV x-ray irradiator. Apoptosis was characterized by immunofluorescence of cleaved caspase 3 and TUNEL assay. DNA damage and repair was determined by immunofluorescence of γH2Ax foci and 53BP1.While cerebral organoids demonstrated no gross morphologic changes to high single dose radiation up to 40 Gy even when incubated for several months following treatment, individual apoptotic cells were readily apparent at 72 hours after 20 Gy. High levels of γH2Ax foci and 53BP1 were observed within hours. While characterization of the kinetics of DNA repair showed significant resolution of DNA damage foci by 24 hours, many foci persisted at 72 hours alongside activation of apoptotic pathways. Choroid plexus tissue was highly resistant to radiation-induced apoptosis.Cerebral organoids provide a viable experimental platform to study effects of radiation in normal CNS tissues, and may potentially aid in the development of neuroprotective strategies.