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. 2016 Mar 1;25(5):989-1000.
doi: 10.1093/hmg/ddv637. Epub 2016 Jan 5.

Assessing Similarity to Primary Tissue and Cortical Layer Identity in Induced Pluripotent Stem Cell-Derived Cortical Neurons Through Single-Cell Transcriptomics

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Free PMC article

Assessing Similarity to Primary Tissue and Cortical Layer Identity in Induced Pluripotent Stem Cell-Derived Cortical Neurons Through Single-Cell Transcriptomics

Adam E Handel et al. Hum Mol Genet. .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
Validation of cortical neuronal phenotype. (A) RT-qPCR showing a reduction in NANOG expression (top) and increased expression of cortical identity markers (bottom) following neural induction in AH017-7. Error bars show the standard deviation from technical triplicates. Immunofluorescence microscopy for: (B) TUJ1 (green) and Synaptophysin (red), scale bar = 10 μm; (C) TUJ1 (green) and TBR1 (red), scale bar = 50 μm; (D) TUJ1 (green) and CUX1 (red), scale bar = 50 μm; all images are also co-stained with DAPI (blue). (E) Repetitive firing evoked by current clamp protocol. (F) Spontaneous electrical activity.
Figure 2.
Figure 2.
Single-cell RT-qPCR data. (A) Heat map of single-cell gene expression data. Cells are shown in rows and genes in columns. The vertical colour key beside the dendrogram indicates the experiment [red = AH017-3 (Repeat 1), blue = AH017-7, green = NHDF1, yellow = AH017-3 (Repeat 2), purple = AH017-3 (Repeat 3) and orange = AH017-7 (Repeat 2)]. The horizontal colour key indicates the functional category of genes. Cells are clustered by Euclidean distance. (B) Co-expression heat map of single-cell gene expression data. The shading represents the magnitude of the Pearson correlation coefficient. Clusters of genes relevant to neuronal function (blue) and neocortical identity (red) are indicated by arrows. Genes not detected in bulk RT-qPCR samples were removed prior to analysis.
Figure 3.
Figure 3.
Single-cell RNA-seq in iPSC-derived cortical neurons. (A) PCA of single-cell RNA-seq on iPSCs (red), neural progenitors (yellow), fetal radial glia (green), fetal newborn cortical neurons (21-week post-conception; blue), fetal matured cortical neurons (21-week post-conception plus 3-week culture; purple) and iPSC-derived cortical neurons (black) (20). (B) Mean expression levels of genes demonstrated by Darmanis et al. to distinguish between different types of adult and fetal cells within the CNS (21). (C) PCA of single-cell RNA-seq on neurons from fetal quiescent cells (16- to 18-week post-conception; red), fetal replicating cells (16- to 18-week post-conception; green), adult temporal cortex (21–63 years old; blue) and iPSC-derived cortical neurons (black).
Figure 4.
Figure 4.
Gene ontology enrichment within differentially expressed gene lists. Bar plots of enrichment of gene ontology terms within lists of significantly differentially expressed genes relative to 72-day-old iPSC-derived cortical neurons (n = 16). Blue bars show enrichment for genes with higher expression in iPSC-derived cortical neurons than primary cells, and red bars show enrichment for genes with higher expression in primary cells than iPSC-derived cortical neurons. All enrichments are significant at FDR < 0.05 and are ordered by the magnitude of enrichment.
Figure 5.
Figure 5.
Principal component analysis. (A) Clustering of cells by experiment on single-cell RT-qPCR data. The percentage of total variance accounted for by each principal component is shown beneath each axis. (B) Clustering of cells by cortical layer identity. The percentage of total variance accounted for by each principal component is shown beneath each axis.
Figure 6.
Figure 6.
Cells with mixed cortical layer identity. (A) Heat map of single-cell gene expression data in neurons (n = 380) for deep and upper layer markers. Neurons (rows) are clustered by Euclidean distance. (B) Box plots showing the magnitude of cortical layer marker expression by RT-qPCR in cells expressing both deep and upper layer markers or those with exclusively deep or upper layer identity. (C) The number of cells expressing each cortical layer marker by RT-qPCR. (D–F) Immunofluorescent images of cells co-expressing the deep layer marker TBR1 (red) and the upper layer markers CUX1 (D), BRN2 (E) or SATB2 (F) (green). Immunofluorescent images were from cortical neurons aged >81 days derived from AH017-3 (C and D) or NHDF1 (E) iPSCs. Scale bars represent 20 μm.

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