Elucidating Astrocytic Diversity in Human Cerebral Organoids

Abstract

Astrocytes are vital supporters and modulators of neuronal function in both health and disease. They play a key role in brain development, plasticity and response to injury and degeneration. Their diversity, especially within a given brain region, has so far been largely underappreciated. Recent studies though have hinted at the potential role astrocyte diversity may play in susceptibility to disease, which may inform therapeutic targets. The lack of consensus regarding astrocyte diversity is in part due to the difficulty of modelling the human brain and astrocyte specification in a dish and the significant differences found between mouse and human astrocytes. To overcome these difficulties, cortical organoid slice cultures derived from human embryonic stem cells (hESCs) were generated. The potential for their long-term growth permits the generation of large numbers of glial cells and allowed for a longitudinal assay of astrocytic diversity. This thesis reports the study of newfound astrocytic diversity in human cortical organoids grown at the air-liquid interface (ALI-COs) for up to 495 days in vitro (DIV). It shows that ALI-CO development displays considerable similarities with the human fetal cortex and undergoes significant gliogenesis at 200DIV. In particular, two novel astrocytic populations, AC1 and AC2 are identified and validated through single cell and spatial transcriptomics. Based on newly identified markers from single-cell RNA sequencing (scRNASeq) results, fluorescence-activated cell sorting (FACS)-based isolation of these populations reveal that they differ in morphology, calcium signalling, synaptogenic and synaptic engulfment potential, consistent with their transcriptome. In addition, these two populations show distinct responses to neurodegeneration and physical injury linked to their reactivity status. These results show that the human cortical slice culture system allows for an alternative approach to the study of human brain cell diversity and may provide a useful platform for the establishment of therapeutic strategies for neurodegenerative diseases and selective vulnerability.