Insights for disease modeling from single-cell transcriptomics of iPSC-derived Ngn2-induced neurons and astrocytes across differentiation time and co-culture

Abstract

Background: Trans-differentiation of human-induced pluripotent stem cells into neurons via Ngn2-induction (hiPSC-N) has become an efficient system to quickly generate neurons a likely significant advance for disease modeling and in vitro assay development. Recent single-cell interrogation of Ngn2-induced neurons, however, has revealed some similarities to unexpected neuronal lineages. Similarly, a straightforward method to generate hiPSC-derived astrocytes (hiPSC-A) for the study of neuropsychiatric disorders has also been described.

Results: Here, we examine the homogeneity and similarity of hiPSC-N and hiPSC-A to their in vivo counterparts, the impact of different lengths of time post Ngn2 induction on hiPSC-N (15 or 21 days), and the impact of hiPSC-N/hiPSC-A co-culture. Leveraging the wealth of existing public single-cell RNA-seq (scRNA-seq) data in Ngn2-induced neurons and in vivo data from the developing brain, we provide perspectives on the lineage origins and maturation of hiPSC-N and hiPSC-A. While induction protocols in different labs produce consistent cell type profiles, both hiPSC-N and hiPSC-A show significant heterogeneity and similarity to multiple in vivo cell fates, and both more precisely approximate their in vivo counterparts when co-cultured. Gene expression data from the hiPSC-N show enrichment of genes linked to schizophrenia (SZ) and autism spectrum disorders (ASD) as has been previously shown for neural stem cells and neurons. These overrepresentations of disease genes are strongest in our system at early times (day 15) in Ngn2-induction/maturation of neurons, when we also observe the greatest similarity to early in vivo excitatory neurons. We have assembled this new scRNA-seq data along with the public data explored here as an integrated biologist-friendly web-resource for researchers seeking to understand this system more deeply: https://nemoanalytics.org/p?l=DasEtAlNGN2&g=NES .

Conclusions: While overall we support the use of the investigated cellular models for the study of neuropsychiatric disease, we also identify important limitations. We hope that this work will contribute to understanding and optimizing cellular modeling for complex brain disorders.

Keywords: Alzheimer’s; Astrocytes; Autism; Induced pluripotent stem cells; Neurons; Ngn2; Schizophrenia; Single cell; Transcriptome.

Fig. 1

UMAP plot of our data highlighting the different conditions (A), pairwise correlations of gene expression in bulk and pseudobulk gene expression for the conditions (B), the same UMAP plot highlighting cluster derived with Louvain clustering (C), and compositions of the Louvain clusters in terms of cells from the five conditions (D)

Fig. 2

A MetaNeighbor analysis of our bulk and pseudobulk expression data with in vivo data from two in vivo studies. Ex_Cor, excitatory cortical; HEW, human embryo week; Astro, astrocytes; Oligo, oligodendrocytes; OPC, oligodendrocyte precursor cells; Endo, endothelial. B, C Seurat integration analysis of our hiPSC-N cells with neuronal cells from two in vivo datasets. B our cells colored by condition. C our cells colored by Louvain cluster. Ex_Cor, excitatory cortical; HEW, human embryo week. D, E Seurat integration analysis of our hiPSC-A cells with non-neuronal cell from two in vivo datasets. D Our cells colored by condition. E Our cells colored by Louvain cluster. SMC, smooth muscle cells; VEC, vascular endothelial cells; VLMC, vascular leptomeningeal cells. Astro, astrocytes; Oligo, oligodendrocytes

Fig. 3

Integration analysis of our Ngn2-induced neuronal data with other single-cell data in Ngn2-induced neurons. A UMAP of Seurat-integrated data from our study and two other scRNA-seq studies of Ngn2-induced neurons (Schornig et al. and Lin et al.), colored by cell cluster. B Same UMAP colored by weeks of Ngn2 induction. C Proportion of cells from different time points in each cell cluster. D Cells from individual studies visualized in the same UMAP as in A and B, again colored by weeks of Ngn2-induction. E Expression of marker genes used to delineate diversity in the lineage composition of Ngn2-indced neurons. F MetaNeighbor analysis of in vitro cells and in vivo cell types (both divided by time points and by study: W, weeks of Ngn2 induction or gestational week; HE, human embryo, individual cell lines used are also indicated)

Fig. 4

Correlation matrix of genome-wide pseudo-bulk expression data from hiPSC-A cells, bulk RNA-seq data from additional in vitro iPSC-derived astrocytes (Tcw = Brennand) and in vivo cell types (Fan and Darmanis). iAstro1-4, in vitro iPSC-derived astrocytes; CtxAstro & MbAstro, cultured primary cortical and midbrain astrocytes. Only the 3000 most highly variable genes were used in the calculation of the correlation coefficients