CelltypeR: A flow cytometry pipeline to characterize single cells from brain organoids
Rhalena A. Thomas,
Julien Sirois,
Shuming Li,
Alexandre Gestin,
Ghislaine Deyab,
Valerio E.C. Piscopo,
Paula Lépine,
Meghna Mathur,
Carol X.-Q. Chen,
Vincent Soubannier,
Taylor M. Goldsmith,
Lama Fawaz,
Thomas M. Durcan,
Edward A. Fon
Affiliations
Rhalena A. Thomas
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada; Corresponding author
Julien Sirois
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Shuming Li
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Alexandre Gestin
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; Université Paris-Saclay, 91190 Gif-sur-Yvette, France
Ghislaine Deyab
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada
Valerio E.C. Piscopo
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Paula Lépine
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Meghna Mathur
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Carol X.-Q. Chen
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Vincent Soubannier
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Taylor M. Goldsmith
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Lama Fawaz
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada
Thomas M. Durcan
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Edward A. Fon
Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, QC H3A 2B4, Canada; The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada
Summary: Motivated by the cellular heterogeneity in complex tissues, particularly in brain and induced pluripotent stem cell (iPSC)-derived brain models, we developed a complete workflow to reproducibly characterize cell types in complex tissues. Our approach combines a flow cytometry (FC) antibody panel with our computational pipeline CelltypeR, enabling dataset aligning, unsupervised clustering optimization, cell type annotating, and statistical comparisons. Applied to human iPSC derived midbrain organoids, it successfully identified the major brain cell types. We performed fluorescence-activated cell sorting of CelltypeR-defined astrocytes, radial glia, and neurons, exploring transcriptional states by single-cell RNA sequencing. Among the sorted neurons, we identified subgroups of dopamine neurons: one reminiscent of substantia nigra cells most vulnerable in Parkinson’s disease. Finally, we used our workflow to track cell types across a time course of organoid differentiation. Overall, our adaptable analysis framework provides a generalizable method for reproducibly identifying cell types across FC datasets in complex tissues.
