Derivation and Molecular Characterization of a Morphological Subpopulation of Human iPSC Astrocytes Reveal a Potential Role in Schizophrenia and Clozapine Response

Abstract

Astrocytes are the most abundant cell type in the human brain and are important regulators of several critical cellular functions, including synaptic transmission. Although astrocytes are known to play a central role in the etiology and pathophysiology of schizophrenia, little is known about their potential involvement in clinical response to the antipsychotic clozapine. Moreover, astrocytes display a remarkable degree of morphological diversity, but the potential contribution of astrocytic subtypes to disease biology and drug response has received little attention. Here, we used state-of-the-art human induced pluripotent stem cell (hiPSC) technology to derive a morphological subtype of astrocytes from healthy individuals and individuals with schizophrenia, including responders and nonresponders to clozapine. Using functional assays and transcriptional profiling, we identified a distinct gene expression signature highly specific to schizophrenia as shown by disease association analysis of more than 10 000 diseases. We further found reduced levels of both glutamate and the NMDA receptor coagonist d-serine in subtype astrocytes derived from schizophrenia patients, and that exposure to clozapine only rescued this deficiency in cells from clozapine responders, providing further evidence that d-serine in particular, and NMDA receptor-mediated glutamatergic neurotransmission in general, could play an important role in disease pathophysiology and clozapine action. Our study represents a first attempt to explore the potential contribution of astrocyte diversity to schizophrenia pathophysiology using a human cellular model. Our findings suggest that specialized subtypes of astrocytes could be important modulators of disease pathophysiology and clinical drug response, and warrant further investigations.

Keywords: astrocyte diversity; d-serine; glutamate; hiPSC; transcription.

Fig. 1.

Derivation and characterization of astrocytic subtype population. (A) Outline of the neural differentiation protocol used to derive subtype astrocytes from a hESC and a hiPSC cell line (hPSCs). RNA-seq for comparison between classical and subtype astrocytes was performed on 180 days old hiPSCs only. (B) Differentiation of hESC and hiPSC-derived subtype astrocytes from day 40 to 180. Scale bar: 50 μm. (C) Morphological diversity of derived subtype astrocytes at day 100 of differentiation. Scale bar: 20 μm. (D) Diverse tail-ends of subtype astrocytes. Scale bar: 10 μm. All experiments in B-D were performed three times. (E) Representative images of GFAP-positive classical and subtype astrocytes derived from control hiPSC lines and used for RNA-seq analysis. Scale bar: 20 μm. (F) Principal component analysis (PCA) of three replicates of hiPSC-derived classical and subtype astrocytes based on the top 500 genes with largest expression variance across samples. (G) Computational deconvolution of four major cell types in the human brain. Data is shown as mean ± SD. *Two-sample t-test: P < .05. (H) Volcano plot showing genes with differential expression (DE) in subtype astrocytes compared to classical astrocytes. The dotted line represents FDR <0.1. The five top genes with strongest association are labeled. (I) GO over-representation test of downregulated DE genes. The top 15 significant (FDR < 0.05) GO terms (biological processes) are displayed. (J) GO over-representation test of upregulated DE genes. The top 15 significantly enriched GO terms are shown.

Fig. 2.

Potential role of subtype astrocytes in SCZ. (A) Schematic illustration of the reprogramming and differentiation process of subtype astrocytes derived from HC and SCZ patients. Clozapine responders (R) and nonresponders (NR) are indicated. (B) All HC and SCZ hiPSCs expressed typical pluripotency markers. (C) All HC and SCZ hiPSCs stained positively for GFAP. (D) Intracellular glutamate levels (fold change) in SCZ subtype astrocytes relative to HC astrocytes. Measurements were performed in triplicates. Data is shown as mean ± SD. *Mann–Whitney U test: P < .05. (E) PCA analysis of two HC and five SCZ subtype astrocyte samples for which RNA-seq data was available. The PCA analysis was based on the top 500 genes with largest expression variance. PC: principal component. (F) Computational deconvolution of human brain cell types in HC and SCZ samples. Data shown as mean ± SD. (G) Volcano plot showing significant DE genes (FDR < 0.1, dotted line). Top 5 genes with lowest P-values are labeled. (H) GO analysis of upregulated DE genes. Top 15 enriched (FDR < 0.05) GO terms (biological processes) are shown. (I) GO analysis of downregulated DE genes. Top 15 enriched (FDR < 0.05) GO terms are shown. (J) Top 15 disease terms of the CURATED DisGeNET database (10 370 diseases) associated with the identified SCZ-related DE genes.

Fig. 3.

Potential role of subtype astrocytes in CLZ response. (A) Fold change of d-serine levels (measured as the number of d-serine-positive puncta) relative to HC1 before CLZ treatment. Left: Before CLZ treatment, Right: after CLZ treatment. The experiments were performed three times. Data shown as mean ± SD. Mann–Whitney U test: *P < .05 and **P < 0.01. (B) Expression levels of genes related to glutamate and d-serine metabolism and transport with nominally significant (P < .05) differences in CLZ-R vs. CLZ-NR samples. (C) PCA plots showing partial and complete clustering on CLZ response status across PCs 1–2 (left) and PCs 3–4 (right), respectively. The plots are based on the top 500 genes with largest expression variation across SCZ samples. (D) Global DE analysis between CLZ-R and CLZ-NR samples. Significant DE genes (FDR < 0.1) are labeled. (E) Heatmap of the four DE genes with differential expression in CLZ responders (R) compared to nonresponders (NR). Expression levels are shown as standardized z scores.