title: Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics.
abstract: Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire OX1 3QX, UK, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK.
authors.forename: Adam E
authors.initials: AE
authors.lastname: Handel
authors.affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK.
authors.forename: Satyan
authors.initials: S
authors.lastname: Chintawar
authors.affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Lalic
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, Oxfordshire OX1 3QT, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Whiteley
authors.affiliation: Dunn School of Pathology, University of Oxford, Oxford, Oxfordshire OX1 3RE, UK.
authors.forename: Jane
authors.initials: J
authors.lastname: Vowles
authors.affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK.
authors.forename: Alice
authors.initials: A
authors.lastname: Giustacchini
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire OX3 7BN and.
authors.forename: Karene
authors.initials: K
authors.lastname: Argoud
authors.affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Sopp
authors.affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
authors.forename: Mahito
authors.initials: M
authors.lastname: Nakanishi
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire OX3 7BN and.
authors.forename: Rory
authors.initials: R
authors.lastname: Bowden
authors.affiliation: Dunn School of Pathology, University of Oxford, Oxford, Oxfordshire OX1 3RE, UK.
authors.forename: Sally
authors.initials: S
authors.lastname: Cowley
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, Oxfordshire OX1 3QT, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Newey
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, Oxfordshire OX1 3QT, UK.
authors.forename: Colin
authors.initials: C
authors.lastname: Akerman
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire OX1 3QX, UK.
authors.forename: Chris P
authors.initials: CP
authors.lastname: Ponting
authors.affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK, zameel.cader@ndcn.ox.ac.uk.
authors.forename: M Zameel
authors.initials: MZ
authors.lastname: Cader
mesh_terms: D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D005260:Female; D005333:Fetus; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D055495:Neurogenesis / Q000235:genetics*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D017470:Receptors, Glutamate / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA; D059010:Single-Cell Analysis; D059467:Transcriptome
chemical_list: D015415:Biomarkers; D009419:Nerve Tissue Proteins; D017470:Receptors, Glutamate