182 Purification and Characterization of Human Neural Stem and Progenitor Cells

Abstract

INTRODUCTION: The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. METHODS: We combine high-dimensional flow cytometry with single cell transcriptomics to comprehensively purify and functionally characterize NSPCs from the developing human brain during mid-gestation. We utilize index sorting based on the combinatorial expression of cell-surface markers (immunophenotype) to directly link surface-marker expression to the expressed transcriptomes of single cells, allowing for rigorous validation of sort purity. We further demonstrate the functional properties of each identified cell type in vitro and in vivo. RESULTS: CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain.THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+ THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a novel transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell, which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. CONCLUSIONS: The results presented here are strong proof-of-concept that distinct cell types from the developing brain can be prospectively isolated based on the expression of multiple surface markers. The combined results from scRNA-seq, in vitro differentiation, and in vivo transplantation offers the most rigorous interrogation of NSPCs’ transcriptomic identities and functional behavior. The modular nature of our antibody panel allows for the easy expansion or simplification of the panel, based on the cell-type of interest, thus providing a valuable tool for future investigation of both molecular and functional NSPC heterogeneity.