Abstract 10- 1000-1015
Category: Basic Science
At the end of the session,
participants will be able to:
- To review the prevailing scientific literature on human astrocyte subtypes and their potential relevance in human disease.
- To discuss the use of single nuclear RNA sequencing and cluster analysis for the identification of transcriptomically distinct astrocyte subtypes.
- To demonstrate the process of validation of antibodies against subtype specific markers using immunohistochemistry and immunofluorescence microscopy.
None to disclose
Brenden Joseph is UBC PGY4 Neuropathology Resident
Brenden Joseph1,2, Kaitlin Sullivan3, Mark S. Cembrowski3, Veronica Hirsch-Reinshagen1,2
1Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada
2Division of Neuropathology, Vancouver General Hospital, Vancouver, BC, Canada
3Department of Cellular and Physiological Sciences, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada
Pathologists, Residents, Medical Students
Identification and validation of immunohistochemical antibodies against transcriptomically distinct human hippocampal astrocyte subtypes
Our understanding of astrocyte function has steadily evolved from an early conceptualization as simplistic support cells to a current appreciation for their diverse and critical physiological roles. There is emerging evidence for the existence of distinct astrocyte subtypes with differential genomic expression, spatial localization, morphology, and physiological function. However, little is known about their specialized functions or the role of specific subtypes in human disease. A lack of established subtype-specific immunohistochemical markers represents a significant barrier to further investigation. To address this limitation, we aim to identify transcriptomically distinct human astrocyte subtypes and validate immunohistochemical markers for these. To identify distinct transcriptomic subtypes of human astrocytes, we used single cell nuclear RNA sequencing of 11,204 astrocytes in human hippocampal surgical samples from 5 patients with intractable mesiotemporal epilepsy. Canonical correlation and Seurat cluster analysis identified 6 distinct clusters of astrocytes with unique transcriptomes and revealed several candidate marker genes unique to each cluster. We are currently validating C8orf34 and NRG4 as subtype specific markers for Cluster 5, which was the most transcriptomically unique cluster. The study’s current and future directions include the selection of commercially available antibodies against candidate marker gene products and validation of these subtype-specific antibodies using double- and triple-label immunofluorescence microscopy with other established markers for various CNS cell types. Establishing validated antibodies will lead to the discovery of subtype-specific involvement of astrocytes in human neurological and psychiatric conditions and advancements in our understanding of human astrocyte physiology.