Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics.
Adam E Handel, Satyan Chintawar, Tatjana Lalic, Emma Whiteley, Jane Vowles, Alice Giustacchini, Karene Argoud, Paul Sopp, Mahito Nakanishi, Rory Bowden, Sally Cowley, Sarah Newey, Colin Akerman, Chris P Ponting, M Zameel Cader
Author Information
Adam E Handel: 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.
Satyan Chintawar: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK.
Tatjana Lalic: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK.
Emma Whiteley: Department of Pharmacology, University of Oxford, Oxford, Oxfordshire OX1 3QT, UK.
Jane Vowles: Dunn School of Pathology, University of Oxford, Oxford, Oxfordshire OX1 3RE, UK.
Alice Giustacchini: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK.
Karene Argoud: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire OX3 7BN and.
Paul Sopp: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK.
Mahito Nakanishi: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Rory Bowden: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire OX3 7BN and.
Sally Cowley: Dunn School of Pathology, University of Oxford, Oxford, Oxfordshire OX1 3RE, UK.
Sarah Newey: Department of Pharmacology, University of Oxford, Oxford, Oxfordshire OX1 3QT, UK.
Colin Akerman: Department of Pharmacology, University of Oxford, Oxford, Oxfordshire OX1 3QT, UK.
Chris P Ponting: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire OX1 3QX, UK.
M Zameel Cader: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire OX3 9DS, UK, zameel.cader@ndcn.ox.ac.uk.
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.
