Human iPSC Glial Mouse Chimeras Reveal Glial Contributions to Schizophrenia

Abstract

In this study, we investigated whether intrinsic glial dysfunction contributes to the pathogenesis of schizophrenia (SCZ). Our approach was to establish humanized glial chimeric mice using glial progenitor cells (GPCs) produced from induced pluripotent stem cells derived from patients with childhood-onset SCZ. After neonatal implantation into myelin-deficient shiverer mice, SCZ GPCs showed premature migration into the cortex, leading to reduced white matter expansion and hypomyelination relative to controls. The SCZ glial chimeras also showed delayed astrocytic differentiation and abnormal astrocytic morphologies. When established in myelin wild-type hosts, SCZ glial mice showed reduced prepulse inhibition and abnormal behavior, including excessive anxiety, antisocial traits, and disturbed sleep. RNA-seq of cultured SCZ human glial progenitor cells (hGPCs) revealed disrupted glial differentiation-associated and synaptic gene expression, indicating that glial pathology was cell autonomous. Our data therefore suggest a causal role for impaired glial maturation in the development of schizophrenia and provide a humanized model for its in vivo assessment.

Keywords: astrocyte; childhood-onset schizophrenia; dysmyelination; glia; glial differentiation; iPSC; induced pluripotent stem cell; mouse models; myelin; schizophrenia.

Figure 1. Functional and genomic assessment of schizophrenia-derived glial progenitor cells

This schematic summarizes the steps involved in our analysis of glial progenitor cells derived from individuals with juvenile-onset schizophrenia, compared to GPCs derived from behaviorally-normal controls. The major output data include effects of SCZ origin on in vivo oligodendrocyte maturation and myelination (Figure 2); in vivo astrocyte differentiation and phenotype (Figure 3); in vitro differential gene expression (Figures 4 and 5); and behavioral phenotype of the human glial chimeric host animals (Figure 6). See also Figure S1 and Table S1.

Figure 2. Schizophrenia-derived hGPCs exhibit aberrant dispersal and relative hypomyelination

Human iPSC GPC chimeras were established by neonatal hGPC injection into shiverer hosts and sacrificed at 19 weeks. GPCs derived from a control subject (A) dispersed primarily in the major white matter tracts, whereas (B) SCZ-derived GPCs (15 yo male) showed less white matter residence and more rapid cortical infilitration. C–D, Sagittal sections reveal that callosal myelination by SCZ GPCs (D) was less dense than that by control hGPCs (C). E–F, Higher power images from chimeric mice engrafted with hGPCs from 4 control patients (E) vs. chimeric mice engrafted with hGPCs from 4 different SCZ patients (F). G, MBP luminance confirmed the greater callosal myelination of CTRL GPC-engrafted vs. SCZ GPC-engrafted mice at 19 weeks (means of 4 different SCZ and CTRL patients each, n>3 mice/patient) (p=0.0002, t-test). H, Absolute donor cell densities were lower in SCZ than control hGPC-engrafted corpus callosum (p<0.0001, t-test), as were the densities of olig2⁺ hGPCs and oligodendroglia (I) (p=0.0064, t-test) and transferrin (TFN)⁺ oligodendroglia (J) (p<0.0001, t-test). See also Figure S2.

Figure 3. Astrocytic differentiation is impaired in schizophrenia hGPC chimeric brain

Human iPSC GPC chimeras were established in immunodeficient shiverer hosts and sacrificed at 19 weeks, and astrocytic diffeerntiation assessed. A–B, representative images of the corpus callosum of mice neonatally injected with iPSC GPCs derived from either control (A, line 22) or schizophrenic (B, line 164) subjects (human nuclear antigen, green; glial fibrillary acidic protein, red). A, Control hiPSC GPCs from all tested patients rapidly differentiated as GFAP⁺ astrocytes with dense fiber arrays in both callosal white and cortical gray matter. B, In contrast, SCZ GPCs were slow to develop mature GFAP expression. At 19 weeks, GFAP⁺ astrocyte densities were significantly greater in mice chimerized with control than SCZ-derived GPCs, both as a group (C), and when analyzed line-by-line (D). This was not just a function of less callosal engraftment, as the proportion of human donor cells that developed GFAP and astrocytic phenotype was significantly lower in SCZ- than control GPC-engrafted mice (E). Sholl analysis of individual astroglial morphologies(Sholl, 1953), as imaged in 150 μm sections and reconstructed in 3D by Neurolucida (J), revealed that astrocytes in SCZ hGPC chimeras differed significantly from their control hGPC-derived counterparts, with fewer primary processes (F), less proximal branching (G), and longer distal fibers (H). When the 3-D tracings (J) were assessed by Fan-in radial analysis (MBF Biosciences)(Dang et al., 2014), control astrocytic processes were noted to extended uniformly in all directions, but SCZ astrocyte processes left empty spaces, indicative of a discontiguous domain structure (I). ***p<0.0001, by t-test (C, E, F, H; by 2-way ANOVA in D; **p<0.002 in I; p<0.0001 by non-linear comparison in G. Scale, A–B = 50 μm, J = 25 μm.

Figure 4. Schizophrenia-derived hGPCs suppress glial differentiation-associated gene expression

RNA sequence analysis reveals differential gene expression by SCZ hGPCs. A, Intersection of lists of differentially expressed genes (DEGs) (log2-fold change >1.00, FDR 5%) obtained by comparison of hGPCs derived from 4 different schizophrenia patients, compared to pooled control hGPCs. B, Network representation of functional annotations for the intersection gene list shown in A. In the upper network, green and red nodes represent down- and up-regulated genes, respectively, and white nodes represent significantly associated annotation terms (FDR-corrected p< 0.01; annotation terms include GO:BP, GO:MF, pathways, and gene families, and nodes are sized by degree). Lower network highlights 4 highly interconnected modules identified by community detection. (C) Top annotation terms identified for each module in B. D, Heat map representation of 12 conserved differentially expressed genes that are associated to module 1 (grey in B, 32.4%), which includes annotations related to neurotransmitter receptor and gated channel activity. E, Heat map representation of 15 conserved differentially expressed genes associated to module 2 (orange in B, 28.7%), which comprises annotations related to cell-to-cell signaling and synaptic transmission. F, Heat map representation of 21 conserved differentially expressed genes associated to module 3 (dark blue in B, 28.7%); annotations related to CNS and glial differentiation. G, Heat map representation of 4 conserved differentially expressed genes that are associated to module 4 (light blue in B, 10.2%), with annotations related to myelination and lipid biosynthesis. The absolute expression in heat maps is shown in UQ-normalized, log2-transformed counts (Li et al., 2015). See also Figures S3 and S4, and Table S2.

Figure 5. Impaired glial differentiation-associated gene expression by SCZ hGPCs

The expression of dysregulated genes in SCZ-derived GPCs, as identified by RNA-seq analysis, was assessed by TaqMan Low Density Array (TLDA) RT-qPCR, then compared to that of control hGPCs. Expression data were normalized to GAPDH endogenous control. Mean ddCt values and standard error ranges, calculated from 4 pooled SCZ GPC lines (n = 19) that were individually compared to 3 pooled control GPC lines (n = 10), are shown. The difference in gene expression by SCZ and control hGPCs was assessed by paired t-tests, followed by multiple testing correction by Benjamini-Hochberg (BH) procedure (*** = p <0.01, ** = p < 0.05, * = p <0.1). 48 genes were assessed. 45 genes are shown, excluding the endogenous control and genes that had high proportions of undetermined or unreliable reactions, LRFN1 and NEUROD6. The vast majority of genes were confirmed as dysregulated in SCZ-derived GPCs. Analysis of TLDA data was performed in ExpressionSuite Software version 1.1, supplied by Applied Biosciences. See also Table S3.

Figure 6. Schizophrenia-derived human glial chimeras have significant behavioral abnormalities

A–E, Behavioral tests were performed in mice chimerized with one of 3 SCZ or 3 control hGPC lines, each line from a different patient. 7–20 recipient mice were tested per cell line, males and females equally. A, Prepulse inhibition Normally-myelinated rag1^−/− mice engrafted with SCZ hGPCs had reduced auditory pre-pulse inhibition (PPI) at all volumes of pre-pulse (A). The extent of PPI differed significantly between control (n=13) and SCZ (n=27) hGPC-engrafted animals (p=0.0008 by ANOVA, F=11.76 [1,114]). B. Elevated Plus Maze Left panel, representative heat maps of the cumulated movement of a mouse engrafted with SCZ hGPCs, relative to its matched normal hGPC-engrafted control, in the elevated plus maze, a test designed to assess anxiety, in which preference for enclosed space and avoidance of open height suggests greater anxiety. Right panel, Mice engrafted with hGPCs from 3 SCZ patients (12 implanted mice each, for n=36 mice total) spent more time in the closed maze arms than did control-engrafted mice (n=36, also derived from 3 patients) (p=0.036, 2-tailed t test). C. Sucrose Preference SCZ GPC-engrafted mice were less likely to prefer sweetened water, suggesting relative anhedonia (p=0.02, Mann-Whitney t-test; n=30 mice derived from 3 SCZ lines; n=17 mice from 3 control lines). D. 3-Chamber Socialization Test Mice engrafted with hGPCs were placed into the middle chamber of a box divided into 3 compartments, one holding an empty cage (bottom, “X” in D) and one containing an unfamiliar mouse (top, filled white circle), then video-tracked for 10 minutes. Mice engrafted with SCZ hGPCs (right heat-map) avoided strangers more than did control mice (left heat-map), spending less time with strangers whether analyzed as the proportion of time spent with the stranger mouse relative to the empty cage (left bar graph; p=0.005) or the net amount of time spent with the stranger mouse (right bar graph; p=0.02); 3 SCZ lines, 39 mice; 4 control lines, 52 mice). E. Novel Object Recognition Mice engrafted with SCZ hGPCs showed significantly poorer novel object recognition (p=0.0006; 3 SCZ lines, 19 mice; 3 control lines, 28 mice). F–G. The diurnal activity and sleep patterns of adult mice (70–80 weeks old) engrafted neonatally with either SCZ or CTRL hGPCs were assessed for 72 hrs in closed chambers (Noldus Ethovision), under continuous video recording. F. The average distance traveled in meters/hr over a 72 hr period was calculated and compared between CTRL mice (gray fill, n=8 mice; lines 22 and 17) and SCZ mice (purple fill; n=10, line 52). Time of day is shown as a 24-hour cycle, with the dark phase indicated by gray background shading. The SCZ mice were significantly more active throughout the observation period than CTRL-engrafted mice (p<0.0001, ANOVA, F=19.32 [1,851]. G. Left, Sample heat-maps of one hour of activity during the light phase (16:00 hrs, 2^nd day in box), the normal period of sleep for mice. The control mouse (left), remains inactive for the entire hour, while the SCZ mouse moves about the cage during much of the hour. Right, The SCZ mice exhibited sleep patterns that were fragmented into bouts of shorter duration than their normal hGPC- chimeric controls (p=0.0026 by ANOVA, F=12.08 [1,24]. Means ± SEM; unpaired, two-tailed Welch-corrected t-tests.