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.

This lapse has in large part been due to the lack of animal models of human glial pathophysiology: mouse brains have mouse glia. We therefore asked whether this limitation might be addressed using a novel model of human glial chimeric mice () paired with the development of protocols for generating bipotential astrocyte-oligodendrocyte glial progenitor cells (GPCs) from patient-specific human induced pluripotent stem cells (hiPSCs) (). In these human glial chimeric mouse brains, the majority of resident glia are replaced by human glia and their progenitors (), allowing human glial physiology, gene expression, and effects on neurophysiological function to be assessed in vivo, in live adult mice (). In this study, we used this glial chimeric model to assess the contribution of human glia to the schizophrenic disease phenotype. To this end, we prepared human glial progenitor cells (hGPCs) from iPSCs derived from fibroblasts taken from either juvenile-onset patients with schizophrenia (SCZ) or their normal controls, assessed the differential gene expression of SCZ hGPCs relative to those of normal subjects, and transplanted these cells into immunodeficient neonatal mice to produce patient-specific human glial chimeric mice. The glial chimeric mice were then analyzed regarding the effects of SCZ derivation on astrocytic and oligodendrocytic differentiation in vivo as well as for behavioral phenotype, and the data thereby obtained correlated to disease-associated gene expression ( Figure 1 ).

This schematic summarizes the steps involved in our analysis of glial progenitor cells derived from individuals with juvenile-onset SCZ compared with 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 ).

Patients with schizophrenia are typically characterized by a relative paucity of white matter and often frank hypomyelination (), and a number of both pathological and neuroimaging studies have highlighted deficiencies in both oligodendroglial density and myelin structure in affected patients (), including at the ultrastructural level (). Furthermore, recent studies have emphasized the role of oligodendrocytes in the metabolic support of neurons, suggesting myelin-independent mechanisms whereby oligodendrocytic dysfunction might yield neuronal pathology (). However, despite compelling genetic, cellular, pathological, and radiological studies that have correlated glial and myelin pathology with schizophrenia, many have assumed that clinical hypomyelination among schizophrenics is secondary to neuronal pathology, so the contribution of cell-autonomous glial dysfunction to schizophrenia has not been well studied.

Schizophrenia is a uniquely human disorder whose phylogenetic appearance parallels that of glial evolution, which accelerated with the appearance of hominids (). In particular, astroglial complexity and pleomorphism increased significantly with hominid evolution, which suggests an association between human glial evolution and the development of human-selective neurological disorders, the neuropsychiatric disorders as a case in point. Indeed, a number of both genome-wide association and differential expression studies have highlighted the frequent dysregulation of glia-selective genes, both astrocytic and oligodendrocytic, in schizophrenia (). At the same time, a number of investigators have highlighted the marked differences between humans and rodents in glial gene expression, in contrast to the relatively conserved nature of neuronal gene expression across mammals (). Together, these studies suggest the correlative association of human glial evolution with the phylogenetic appearance of schizophrenia.

As an additional metric of SCZ-associated behavior, we then assessed sleep and diurnal activity patterns of human SCZ and CTRL glial chimeras, directly comparing mice engrafted with either SCZ (line 52) or control (line 22) hGPCs. We found that mice engrafted with SCZ GPCs were significantly more active than control mice engrafted with normal hGPCs. As measured by meters moved per hour, over the course of a 72-hr video recording (Noldus Ethovision), the SCZ hGPC chimeric mice moved significantly more than their normal hGPC-engrafted controls (two-way ANOVA, F = 48.35, p < 0.0001) ( Figure 6 F). Interestingly, although the SCZ-associated increment in activity largely occurred during nighttime periods of wakefulness, the SCZ mice also manifested disrupted sleep patterns, as measured by the duration of bouts of inactivity, a surrogate for electroencephalogram (EEG)-validated sleep ( Figure 6 G). Within the half hour following the phase transition from dark to light (when mice normally sleep), the control (CTRL) mice had more continuous, uninterrupted patterns of sleep, with an average sleep bout of 511.5 ± 36.4 s (8.53 min), whereas SCZ mice were asleep for 306.2 ± 43.7 s, or 5.1 min per bout (p < 0.01 by two-way ANOVA, with Bonferroni post hoc t tests). The shorter average periods of inactivity manifested by SCZ hGPC mice during the normal daytime transition to sleep suggest that SCZ hGPC chimerization disrupted normal daytime sleep patterns while increasing nighttime activity. Together, these results suggest that SCZ glial chimerization was sufficient to yield heightened anxiety and fear in engrafted recipients as well as disease-associated deficits in socialization, cognition, and sleep patterning, all features associated with human SCZ.

We first asked whether schizophrenic derivation of engrafted glia affected prepulse inhibition (PPI), a behavioral hallmark of both clinical schizophrenics and animal models thereof (). PPI reflects the coordination of sensorimotor gating in the CNS, and its diminution may predict aspects of a schizophrenic phenotype (). We found that, when assessed at 6 months of age, the latest time point at which the C57BL/6 background strain of our rag1mice can be reliably assessed (because these mice suffer premature auditory loss that might otherwise diminish auditory PPI), that mice engrafted with SCZ hGPCs exhibited significantly diminished auditory prepulse inhibition ( Figure 6 A) and did so at all volumes of prepulse. Given the strong effect of SCZ glial chimerization on PPI, we next asked whether SCZ glial chimerization might be associated with changes in behavior in cognitive and socialization tests. To that end, we compared SCZ and control chimeras in a battery of behavioral tests that included the elevated plus maze, a measure of anxiety (); the three-chamber social challenge (); novel object recognition, a focused measure of executive memory (); and the preference for sucrose water, a test for anhedonia (). In each, mice chimerized with one of three SCZ or three control patient-derived lines were compared; each line was derived from a different patient. Between 6–12 recipient mice were engrafted and tested per cell line, or 17–36 mice per group for each behavioral comparison, with a typically equal balance of male and female recipients. These animals were tested beginning between 30–36 weeks of age, and testing typically lasted 3 weeks. Over the tested age range, the SCZ GPC chimeric mice exhibited a number of significant differences in behavior relative to their control hGPC-engrafted counterparts. SCZ hGPC mice exhibited greater avoidance of the open arms in the elevated plus maze than the normal hGPC-engrafted controls (n = 36 mice/group, each including 12 mice engrafted with hGPCs from each of three patients; p = 0.036, two-tailed t test), suggesting that the SCZ hGPC mice were prone to higher anxiety when challenged ( Figure 6 B). In addition, the SCZ hGPC mice showed less preference for sucrose water, consistent with relative anhedonia ( Figure 6 C), less interest in strange mice in the three-chamber social test ( Figure 6 D), and relatively poor novel object recognition ( Figure 6 E), reflecting relative impairment in executive memory.

(G) Left: Sample heatmaps of 1 hr of activity during the light phase (16:00, second day in box), the normal period of sleep for mice. The control mouse (left) remains inactive for the entire hour, whereas the SCZ mouse moves about the cage during much of the hour. Right: 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.

(F) The average distance traveled in meters/hour 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-hr cycle, with the dark phase indicated by gray background shading. SCZ mice were significantly more active throughout the observation period than CTRL-engrafted mice (p < 0.0001, ANOVA, F = 19.32 [1,851].

(F and 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 hr in closed chambers (Noldus Ethovision) under continuous video recording.

(D) Three-chamber socialization test. Mice engrafted with hGPCs were placed into the middle chamber of a box divided into three compartments, one holding an empty cage (bottom, X) and one containing an unfamiliar mouse (top, filled white circle) and then video-tracked for 10 min. Mice engrafted with SCZ hGPCs (right heat-map) avoided strangers more than control mice (left heatmap), spending less time with strangers, whether analyzed as the proportion of time spent with the strange mouse relative to the empty cage (left bar graph, p = 0.005) or the net amount of time spent with the strange mouse (right bar graph, p = 0.02); three SCZ lines, 39 mice; four control lines, 52 mice).

(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 three SCZ lines; n = 17 mice from three control lines).

(B) Elevated plus maze. Left: representative heatmaps 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: mice engrafted with hGPCs from three SCZ patients (12 implanted mice each for n = 36 mice total) spent more time in the closed maze arms than control-engrafted mice (n = 36, also derived from three patients) (p = 0.036, two-tailed t test).

(A) Prepulse inhibition normally myelinated rag1 −/− mice engrafted with SCZ hGPCs had reduced auditory prepulse inhibition (PPI) at all volumes of prepulse. 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]).

(A–E) Behavioral tests were performed in mice chimerized with one of three SCZ or three control hGPC lines, each line from a different patient. 7–20 recipient mice were tested per cell line, males and females equally.

The use of the elevated plus maze as an assay of anxiety-related behavior in rodents.

We next asked whether the alterations in glial distribution and differentiation observed in mice engrafted with SCZ hGPCs might alter the behavioral phenotype of the host mice. In particular, we postulated that the aberrant infiltration of hGPCs and their derived astroglia into the developing cortex might influence information processing within the cortex when mature. As noted, past studies have reported both the influence of astrocytic networks on synaptic efficacy and plasticity and the differential competence of hominid glia in this respect (). Human glial chimeric mice manifest a lower threshold for hippocampal long-term potentiation (LTP) and learn more rapidly, with superior performance in a variety of learning tasks that include auditory fear conditioning, novel object and place recognition, and Barnes maze navigation. In each of these tests, but not in any test of social interactivity or primary perception, human glial chimeras acquire new causal associations more quickly than allografted or untransplanted controls (). Thus, engrafted human GPCs and their daughter glia can integrate into, and substantially modify, developing neural networks (). On that basis, we postulated that the disruption in normal glial development noted in our SCZ glial chimeras might yield disease-associated changes in learning and behavior. To address this question, we assessed the behavioral phenotypes of immunodeficient but otherwise wild-type mice neonatally engrafted with SCZ GPCs relative to matched hosts engrafted with control-derived GPCs. For these experiments, we used normally myelinated hosts rather than shiverer mice to produce mice chimeric only for human GPCs and astrocytes and not for oligodendroglia, thus isolating any observed behavioral effects to SCZ hGPCs and astrocytes.

Together, these data suggest the importance of glia-associated synaptic gene expression in SCZ and emphasize the heterogeneity of pathways that might be mechanistically complicit in its dysregulation. These data also highlight the point that, although the neuronal localization of these synaptic proteins has long been recognized, their synthesis by glia and the synaptic contributions thereof have not been specifically discussed, although cell-type-specific transcriptional databases have noted significant glial expression of these genes (). Because NRXN1, a synapse-associated transcript closely linked to SCZ (), was one of the most strongly and consistently downregulated glial genes across our patients, we verified the downregulation of its expression by SCZ glia by immunoblotting CD140a-sorted, neuron-free isolates of SCZ and control hGPCs. Western blots revealed that neurexin-1 was indeed abundantly expressed by human GPCs and that neurexin-1 protein levels were sharply lower in otherwise matched SCZ hGPCs ( Figure S5 ).

These expression data suggest that the diminished myelination of SCZ hGPC-transplanted shiverer brains reflected aberrant oligodendrocytic differentiation from the engrafted SCZ hGPCs. Similarly, because hGPCs give rise to astrocytes as well as oligodendrocytes, the RNA expression data suggest an analogous impediment to astrocytic differentiation. The functional consequences of the latter are especially profound, given the critical role of astrocytes in synaptic development and function; indeed, the relative suppression of astrocytic differentiation by SCZ hGPCs suggests a glial contribution to the impaired synaptic function noted in SCZ. In that regard, further functional analysis of SCZ-associated dysregulated hGPC genes identified channel and receptor activity as well as synaptic transmission as the most differentially affected functions besides glial differentiation ( Figures 4 D and 4E). These disease-linked channel and synapse-associated genes were largely downregulated in SCZ hGPCs and included a number of potassium channel genes ( Figure 4 D), including KCND2, KCNJ9, KCNK9, and KCNA3, as well as a number of transcripts associated with synaptic development and function ( Figure 4 E; Table S2 ). The latter included NXPH1, NLGN3, and LINGO1, among others ( Table S3 Figures S3 and S4 ), synaptic genes whose dysregulation has been previously linked to both SCZ and autism spectrum disorders (). Although the expression of these latter genes was suppressed in hGPCs derived from all four SCZ patients, other synapse-associated genes, such as NRXN1, NLGN1, DSCAML1, and the SLITRKs 2–5, were sharply downregulated in hGPCs derived from three of the four patients but not in the fourth ( Table S3 ). However, other synapse-associated transcripts, like NXPH3 and NTRNG2, were similarly downregulated in some patients but not others. TaqMan low-density arrays were used for quantitative real-time PCR validation of these and other dysregulated transcripts of interest and confirmed the significant differential downregulation of these differentiation- and synaptic function-associated genes ( Figure 5 ).

The expression of dysregulated genes in SCZ-derived GPCs, as identified by RNA-seq analysis, was assessed by TaqMan low-density array (TLDA) qRT-PCR and then compared with that of control hGPCs. Expression data were normalized to the GAPDH endogenous control. Mean delta delta Ct (ddCt) values and SE ranges, calculated from four pooled SCZ GPC lines (n = 19) that were individually compared with three 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.

To better define the molecular basis for the apparent impediment to terminal glial differentiation in SCZ GPC-engrafted mice and to define which aspects of that deficit might be cell-autonomous, we next used RNA sequencing (RNA-seq) analysis to identify the differentially expressed genes of SCZ iPSC-derived GPCs relative to those of control-derived glia. We used sequencing data to reconstruct the transcriptional patterns of hGPCs derived from four different SCZ and three control patients. hGPCs were derived at time points ranging from 154–242 days in vitro and sorted for hGPCs using CD140a-targeted fluorescence-activated cell sorting (FACS). Using a 5% false discovery rate (FDR) and a fold change threshold of 2, we identified a total of 116 mRNAs that were differentially expressed by CD140a-sorted SCZ hGPCs relative to their control iPSC hGPCs ( Figures 4 A and 4B ). Among the genes most differentially expressed by CD140a-sorted SCZ hGPCs were a host of glial differentiation-associated genes, in particular those associated with early oligodendroglial and astroglial lineage progression, which were uniformly downregulated in the SCZ hGPCs relative to their normal controls ( Figures 4 C and 4F). These included a coherent set of the key GPC lineage transcription factors OLIG1, OLIG2, SOX10, and ZNF488 as well as genes encoding stage-regulated proteins involved in myelination, such as GPR17, UGT8, OMG, and FA2H ( Figure 4 G; see Table S2 and Figures S3 and S4 for detailed gene expression data).

(G) Heatmap representation of four conserved DEGs that are associated with module 4 (light blue in B, 10.2%), with annotations related to myelination and lipid biosynthesis. The absolute expression in heatmaps is shown in UQ-normalized, log2-transformed counts ().

(D) Heatmap representation of 12 conserved DEGs that are associated with module 1 (gray in B, 32.4%), which includes annotations related to neurotransmitter receptor and gated channel activity.

(B) Network representation of functional annotations for the intersection gene list shown in (A). In the top network, green and red nodes represent down- and upregulated genes, respectively, and white nodes represent significantly associated annotation terms (FDR-corrected p < 0.01; annotation terms include Gene Ontology: Biological Process (GO:BP), Gene Ontology: Molecular Function (GO:MF), pathways, and gene families, and nodes are sized by degree). The bottom network highlights four highly interconnected modules identified by community detection.

(A) Intersection of lists of differentially expressed genes (DEGs) (log2 fold change > 1.00, FDR 5%) obtained by comparison of hGPCs derived from four different SCZ patients compared with pooled control hGPCs.

We next asked whether the SCZ hGPCs that prematurely entered the gray matter differentiated instead into astrocytes in that environment or whether they rather manifested an impairment in lineage progression that prevented their astrocytic differentiation as well. Both SCZ- and control hGPC-engrafted shiverer brains were immunostained for astrocytic glial fibrillary acidic protein (GFAP) 19 weeks after neonatal graft using a species-specific anti-human GFAP antibody. We found that astrocytic maturation from engrafted hGPCs was markedly deficient in the SCZ hGPC-engrafted brains (n = 19, derived from 3 SCZ patient lines, and n = 12 control mice, from 3 control patients) ( Figures 3 A and 3B ). In the callosal white matter as well as in both the striatal and cortical gray matter, astrocytic differentiation by SCZ hGPCs was significantly less than that of control GPCs so that, although all control hGPC forebrains showed dense human GFAPastrocytic maturation, far fewer SCZ hGPCs manifested hGFAP expression and an astrocytic phenotype (controls: 6,616 ± 672.3 GFAPcells/mmin callosum, n = 12; SCZ: 1,177 ± 276.6 GFAPcallosal cells/mm, n = 19; p < 0.0001 by two-tailed t test; Figure 3 C). This defect in astrocytic differentiation was consistently observed in all mice (n = 19) derived from the three SCZ patients assessed compared with the control GPC-engrafted mice (n = 12) derived from three normal subjects ( Figure 3 D) and reflected in part the lower proportion of GFAPastrocytes that developed among engrafted human cells in the SCZ HGPC-engrafted brains ( Figure 3 E). Furthermore, Sholl analysis of individual astroglial morphologies (), as imaged in 150-μm sections and reconstructed in Neurolucida ( Figure 3 J), revealed that astrocytes in SCZ hGPC chimeras differed significantly from their control hGPC-derived counterparts, with fewer primary processes ( Figure 3 F), less proximal branching ( Figure 3 G), longer distal fibers ( Figure 3 H), and less coherent domain structure ( Figure 3 I). Thus, SCZ hGPCs derived from multiple patients exhibited a common defect in phenotypic maturation and, hence, proved to be deficient in astrocytic differentiation as well as myelination.

∗∗∗ p < 0.0001 by t test (C, E, F, and H) and by two-way ANOVA (D); ∗∗ p < 0.002 (I); p < 0.0001 by non-linear comparison (G). Scale bars, 50 μm (A and B) and 25 μm (J).

(F–J) Sholl analysis of individual astroglial morphologies (), 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 3D tracings (J) were assessed by Fan-in radial analysis (MBF Biosciences;), an approach by which 3D fiber distributions are quantified as to radial segments occupied, control astrocytic processes were noted to extend uniformly in all directions, whereas SCZ astrocytic processes left empty spaces, indicative of a discontiguous domain structure (I).

(C–E) 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 because the proportion of human donor cells that developed GFAP and an astrocytic phenotype was significantly lower in SCZ- than control GPC-engrafted mice (E).

(A and 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). Control hiPSC GPCs from all tested patients rapidly differentiated as GFAP + astrocytes, with dense fiber arrays in both callosal white and cortical gray matter (A). In contrast, SCZ GPCs were slow to develop mature GFAP expression (B).

Because the SCZ hGPC-engrafted shiverers manifested deficient myelination, we asked whether this was due to a relative failure of SCZ hGPCs to remain within white matter or, rather, due to a cell-intrinsic failure in myelinogenesis. Examining 19-week-old SCZ and control hGPC-engrafted shiverer mice, we found significantly fewer human nuclear antigen (hNA)-defined, donor-derived cells in SCZ hGPC-engrafted shiverer white matter (40,615 ± 2,189 × 10hNAcells/mm, n = 18) than in mice identically transplanted with control hGPCs (69,970 ± 4,091/mm, n = 32, p < 0.0001 by two-tailed t test;) ( Figure 2 H). Moreover, the numbers of hNAdonor cells co-expressing the oligodendroglial lineage marker Olig2 were similarly depressed in SCZ hGPC-engrafted mice (33,619 ± 2,435/mm, n = 26) relative to control hGPC-engrafted mice (46,139 ± 2,858/mm, n = 17, p < 0.002 by two-tailed t test) ( Figure 2 I). On that basis, we next found that the density of transferrin-defined human oligodendroglia was similarly lower in the callosal white matter of SCZ hGPC chimeras than in control hGPC chimeras (8,778 ± 892.2/mm, n = 25 versus 17,754 ± 2,023/mm, n = 17, respectively; p = 0.0006 by two-tailed t test) ( Figure 2 J). These data indicate that SCZ GPCs are deficient not only in their colonization of the forebrain white matter but also in their oligodendrocytic differentiation, with a resultant suppression of central myelinogenesis. Together, these findings suggest that SCZ hGPCs migrate aberrantly, traversing rather than homing to developing white matter, thus yielding relatively poor white matter engraftment, deficient myelin formation, and premature cortical entry relative to normal GPCs.

We first noted that the SCZ hGPCs manifested an aberrant pattern of migration upon neonatal transplantation. Normal control hGPCs invariably expanded through the white matter before colonizing the cortical gray matter ( Figure 2 A), as we have noted previously in both fetal tissue- and hiPSC GPC-engrafted shiverer mice (). In contrast, SCZ GPCs preferentially migrated earlier into the gray matter in shiverer mice, with large numbers traversing without stopping in the callosal white matter (n = 4 lines from 4 different patients, each with ≥3 mice/patient, each versus paired controls) ( Figure 2 B; Figure S2 ). This resulted in significantly fewer donor hGPCs in the white matter of shiverers engrafted with SCZ GPCs ( Figures 2 H and 2I; Figure S2 ). Importantly, this was associated with substantially diminished central myelination in these mice, as reflected by both MBP immunostaining ( Figures 2 C–2F) and myelin luminance ( Figure 2 G).

We first asked whether SCZ hGPCs differed from wild-type hGPCs in their myelination competence. To this end, we implanted SCZ hGPCs into neonatal immunodeficient shiverer mice (rag2× MBP), a congenitally hypomyelinated mutant lacking myelin basic protein (MBP) (). As these otherwise myelin-deficient mice matured, their engrafted hGPCs differentiated into both astrocytes and myelinogenic oligodendrocytes, yielding mice chimeric for individual patient-derived glia (). By this means, we established mice with patient-specific, largely humanized forebrain white matter derived from SCZ or control subjects ( Figures 2 A–2D).

p < 0.002 (I);p < 0.0001 (H, J) or <0.0002 (G). In (C) and (D), the figures are derived from individual higher power photos taken in the X-Y plane, then stitched together as wide-field montages in StereoInvestigator. See also Figure S2

(H–J) Absolute donor cell densities were lower in SCZ- than control hGPC-engrafted corpus callosum (H, p < 0.0001, t test), as were the densities of olig2 + hGPCs and oligodendroglia (I, p = 0.002, t test) and transferrin (TFN) + oligodendroglia (J, p < 0.0001, t test).

(G) MBP luminance confirmed the greater callosal myelination of CTRL GPC-engrafted versus SCZ GPC-engrafted mice at 19 weeks (means of four different SCZ and CTRL patients each, n ≥ 3 mice/patient) (p = 0.0002, t test).

(E and F) Higher-power images from chimeric mice engrafted with hGPCs from four control patients (E) versus chimeric mice engrafted with hGPCs from four different SCZ patients (F).

(C and D) Sagittal sections reveal that callosal myelination by SCZ GPCs (D) was less dense than that by control hGPCs (C).

(A and B) GPCs derived from a control subject (A) dispersed primarily in the major white matter tracts, whereas SCZ-derived GPCs (B, 15-year-old male) showed less white matter residence and more rapid cortical infiltration.

Patients with juvenile-onset SCZ as well as healthy young adult controls free of known mental illness were recruited, and skin biopsies were obtained from each. Patient identifiers were not available to investigators besides the treating psychiatrist, although age, gender, race, diagnosis, and medication history accompanied cell line identifiers. Fibroblasts were isolated from each sample; from these, 11 new independent hiPSC lines were derived from eight patient samples (five juvenile-onset SCZ patients and three healthy gender-matched and age-analogous controls ( Table S1 ). iPSCs were generated using the excisable floxed polycistronic hSTEMCCA lentivirus () encoding Oct4, Sox2, Klf4, and c-Myc (). All lines were initially characterized and validated as pluripotent using global transcriptome profiling by RNA sequencing to assess pluripotent gene expression as well as immunostaining for Oct4, Nanog, and SSEA4. The identity of each iPSC line was confirmed to match the parental donor fibroblasts using short tandem repeat (STR)-based DNA fingerprinting. iPSC line isolates were also karyotyped concurrently with these experiments to confirm genomic integrity. An additional well-characterized hiPSC control line, C27 (), was used to ensure that our control engraftment and differentiation data were consistent with prior studies in our lab (). Altogether, we evaluated hGPC preparations from seven iPSC lines derived from five SCZ patients and five iPSC lines derived from four control subjects ( Table S1 ). We instructed these iPSC cells to a GPC fate as described previously () and, after ≥105 days in vitro (DIV) under glial differentiation conditions, validated the predominant GPC phenotype of each cell population using flow cytometry for CD140a/PDGFαR (platelet-derived growth factor receptor alpha) ( Figure S1 ). To optimize glial differentiation in vivo, we limited transplants to preparations in which most cells were CD140aGPCs, with the remainder astroglial.

Discussion

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Bergles D.E. Synaptic signaling between GABAergic interneurons and oligodendrocyte precursor cells in the hippocampus. Besides the anatomic observation of deficient astrocytic maturation in SCZ hGPC chimeras, our genomic analysis of SCZ-derived hGPCs revealed significant downregulation in hGPCs derived from all four SCZ patients of a number of synaptic genes, including neuroligin-3, neuroexophilin-1, and LINGO1, relative to their normal controls ( Table S3 Figure 5 ). Other synapse-associated genes, such as neurexin-1 and DSCAML1, were significantly and sharply downregulated in GPCs derived from three patients (lines 8, 29, and 51) but not in the fourth (line 164). Similarly, SLITRKs 2–5 were significantly and sharply downregulated in GPCs derived from three patients (lines 8, 51, and 164) but not in a fourth (line 29), which was instead associated with sharp downregulation of LINGO1, DSCAML1, and several neurexins and neuroexophilins; these data suggest heterogeneity of transcriptional dysfunction that may lead to a final common pathway of glia-involved synaptic dysfunction in SCZ ( Tables S2 and S3 ). These transcripts are critical contributors to synaptic stabilization and function () but, although typically considered neuronal, may be produced significantly by glial cells as well (). The relative downregulation of these genes by SCZ hGPCs may reflect the suppression of mature glial transcripts in these cells, coincident with their relative block in glial differentiation. This, in turn, may lead to a relative failure of SCZ hGPCs and their derived astrocytes to provide these key proteins to their neuronal partners as well as a potential failure on the part of glial progenitors receiving synaptic inputs to respond to afferent stimulation (). Thus, besides the structural havoc that might be expected of a cortical connectome formed without normal astrocytic support, the synaptic structure of the resultant networks might be expected to be destabilized by poor SCZ glial provision to the synaptic cleft of key astrocytic proteins required for normal synaptic maintenance and function.

Windrem et al., 2008 Windrem M.S.

Schanz S.J.

Guo M.

Tian G.F.

Washco V.

Stanwood N.

Rasband M.

Roy N.S.

Nedergaard M.

Havton L.A.

et al. Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Windrem et al., 2014 Windrem M.S.

Schanz S.J.

Morrow C.

Munir J.

Chandler-Militello D.

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Goldman S.A. A competitive advantage by neonatally engrafted human glial progenitors yields mice whose brains are chimeric for human glia. Wang et al., 2013 Wang S.

Bates J.

Li X.

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Goldman S.A. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. SCZ is genetically heterogeneous, so anatomic and behavioral pathology may vary significantly among animals chimerized with GPCs derived from different patients. It is thus critical that the results obtained from chimeras established with control hiPSC GPCs be stable across both distinct lines of donor cells and among recipient mice. The chimeric brains established from the hGPCs of three different SCZ patients were thus compared anatomically with those established from GPCs derived from three control patients. None of the controls manifested the white matter-avoidant dispersal pattern of the SCZ hGPC chimeras. Similarly, we never noted this pattern of SCZ hGPC avoidance of the white matter in any of several hundred human glial chimeras engrafted in other studies with either fetal tissue-derived () or normal iPSC-derived () hGPCs.