Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics

Human Molecular Genetics, 2016, Vol. 25, No. 5 989–1000 doi: 10.1093/hmg/ddv637 Advance Access Publication Date: 5 January 2016 Original Article ORIGINAL ARTICLE Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics 1 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, 3Department of Pharmacology, University of Oxford, Oxford, Oxfordshire OX1 3QT, UK, 4Dunn School of Pathology, University of Oxford, Oxford, Oxfordshire OX1 3RE, UK, 5Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire OX3 7BN and 6Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan 2 *To whom correspondence should be addressed. Tel: +441865222656; Fax: +441865222737; Email: 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. Singlecell 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 iPSCderived 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. † Co-first authors. Received: September 1, 2015. Revised and Accepted: December 31, 2015 © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 989 Adam E. Handel1,2,†, Satyan Chintawar2,†, Tatjana Lalic2, Emma Whiteley3, Jane Vowles4, Alice Giustacchini2, Karene Argoud5, Paul Sopp2, Mahito Nakanishi6, Rory Bowden5, Sally Cowley4, Sarah Newey3, Colin Akerman3, Chris P. Ponting1 and M. Zameel Cader2, * 990 | Human Molecular Genetics, 2016, Vol. 25, No. 5 Introduction Results Single-cell RT-qPCR neuronal identity We generated cortical neurons using a well-established protocol with small molecule dual SMAD inhibition for neural induction followed by plating of neuroepithelial cells for final differentiation (2). Over the course of neuronal differentiation, cultures showed the expected decrease in expression of pluripotency genes and Comparison with single-cell RNA-seq In order to assess how well the transcriptomes of our iPSC-derived cortical neurons reflected those of single neurons from human fetal or adult neurons, we generated single-cell RNAseq data from sixteen 72-day-old iPSC-derived cortical neurons. Single-cell gene expression estimates from single-cell whole transcriptomics correlated well with our previous single-cell multiplex RT-qPCR data (r = 0.84, P < 10−16; Supplementary Material, Fig. S4). Single iPSC-derived neurons clustered closely with fetal but not adult cortical neurons and expressed high levels of genes specific for post-mitotic fetal neurons (Fig. 3A–C; Supplementary Material, Fig. S5) (20,21). Investigating the cellular basis of neurological diseases, especially those affecting the central nervous system (CNS), is rendered particularly challenging by the inaccessibility of the tissues involved. Induced pluripotent stem cell (iPSC)-based models have the potential to allow in vitro investigation of these tissues in human samples from patients affected by such diseases and, importantly, how disease progresses over time (1). Protocols have been developed capable of generating cortical cells from human iPSCs, which appear to adopt specific cortical layer identities and develop functional synapses (2–6). Most transcriptomic studies of iPSC-derived cortical neurons have examined expression in samples pooled from a whole population of cells so would miss potential cell type-specific or layer-specific effects (7,8). The development of single-cell gene expression platforms, such as microfluidic chips, as well as evolving chip-free single-cell RNA-seq technologies, make such studies a viable method to investigate iPSC-derived cortical neuron cultures at single-cell resolution (9,10). This has the advantage that the relative abundance of different cell types may be discerned, and so comparisons between iPSC-derived and primary tissues can be made at the level of individual cells. A core set of cortical layer markers has been used within the stem cell research community to establish the presence of neurons with different layer identities in iPSC-derived cortical neuronal cultures (2,4,11). However, many of these markers were inferred from studies of mouse brain or immunohistochemistry of human fetal brain, so the robustness of such markers in assigning layer identity to single neurons by single-cell transcriptomics approaches is unknown (12,13). The degree of heterogeneity present in cortical neurons derived from iPSCs is a critically important aspect of in vitro models to understand. Layer-specific and phenotypic cellular identity is particularly relevant prior to applying such models to address disease-specific hypotheses. Cortical neurons derived from iPSCs using such methods have been used to study a wide variety of neurodevelopmental and neurodegenerative conditions, and recapitulate disease-relevant phenotypes (1). In the case of Alzheimer’s disease, iPSC-derived cortical neurons displayed aberrant Aβ secretion and (...truncated)