GLIA PRECURSOR CELLS FOR USE IN THE TREATMENT OF SCHIZOPHRENIA

Description

FIELD

[0001] The claimed invention relates to a preparation of glial progenitor cells for use in treating Schizophrenia.BACKGROUND

[0002] There are a number of uniquely human neurological disorders, whose phylogenetic appearance parallels that of human glial evolution, which accelerated with the appearance of hominids (Oberheim et al., "Astrocytic Complexity Distinguishes the Human Brain," Trends in Neurosciences 29:1-10 (2006); Oberheim et al., "Uniquely Hominid Features of Adult Human Astrocytes," The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 29:3276-3287 (2009); Horrobin, D. F., "Schizophrenia: The Illness That Made Us Human," Med Hypotheses 50:269-288 (1998)). 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. Indeed, a number of both genome-wide association and differential expression studies have highlighted the frequent dysregulation of glial-selective genes, both astrocytic and oligodendrocytic, in, for example, schizophrenia (Walsh et al., "Rare Structural Variants Disrupt Multiple Genes in Neurodevelopmental Pathways in Schizophrenia," Science 320:539-543 (2008); Aberg et al., "Human QKI, A Potential Regulator of mRNA Expression of Human Oligodendrocyte-Related Genes Involved in Schizophrenia," Proceedings of the National Academy of Sciences of the United States of America 103:7482-7487 (2006); Roy et al., "Loss of erbB Signaling in Oligodendrocytes Alters Myelin and Dopaminergic Function, A Potential Mechanism for Neuropsychiatric Disorders," Proceedings of the National Academy of Sciences of the United States of America 104:8131-8136 (2007); Takahashi et al., "Linking Oligodendrocyte and Myelin Dysfunction to Neurocircuitry Abnormalities in Schizophrenia," Prog Neurobiol 93:13-24 (2011); Georgieva et al., "Convergent Evidence That Oligodendrocyte Lineage Transcription Factor 2 (OLIG2) and Interacting Genes Influence Susceptibility to Schizophrenia," Proceedings of the National Academy of Sciences of the United States of America 103:12469-12474 (2006); Hof et al., "Molecular and Cellular Evidence for an Oligodendrocyte Abnormality in Schizophrenia," Neurochem Res 27:1193-1200 (2002); Hakak et al., "Genome-Wide Expression Analysis Reveals Dysregulation of Myelination-Related Genes in Chronic Schizophrenia," Proceedings of the National Academy of Sciences of the United States of America 98:4746-4751 (2001)).

[0003] Patients with schizophrenia are typically characterized by a relative paucity of white matter and often frank hypomyelination (Takahashi et al., "Linking Oligodendrocyte and Myelin Dysfunction to Neurocircuitry Abnormalities in Schizophrenia," Prog Neurobiol 93:13-24 (2011); Connor et al., "White Matter Neuron Alterations in Schizophrenia and Related Disorders," International Journal of Developmental Neuroscience : The Official Journal of the International Society for Developmental Neuroscience 29:325-334 (2011); McIntosh et al., "White Matter Tractography in Bipolar Disorder and Schizophrenia," Biological Psychiatry 64:1088-1092 (2008); Maniega et al., "A Diffusion Tensor MRI Study of White Matter Integrity in Subjects at High Genetic Risk of Schizophrenia," Schizophrenia Research 106:132-139 (2008); Fields, R. D., White Matter in Learning, Cognition and Psychiatric Disorders," Trends in Neurosciences 31:361-370 (2008); Gogtay et al., "Three-Dimensional Brain Growth Abnormalities in Childhood-Onset Schizophrenia Visualized by Using Tensor-Based Morphometry," Proceedings of the National Academy of Sciences of the United States of America 105:15979-15984 (2008)). A number of both pathological and neuroimaging studies have highlighted deficiencies in both oligodendroglial density and myelin structure in affected patients (Fields, R. D., White Matter in Learning, Cognition and Psychiatric Disorders," Trends in Neurosciences 31:361-370 (2008); Xia et al., "Behavioral Sequelae of Astrocyte Dysfunction: Focus on Animal Models of Schizophrenia," Schizophrenia Research (2014); Rapoport et al., "The Neurodevelopmental Model of Schizophrenia: Update 2005," Molecular Psychiatry 10:434-449 (2005); Langmead et al., "Fast Gapped-Read Alignment with Bowtie 2," Nature Methods 9:357-359 (2012)), including at the ultrastructural level (Uranova et al., "Ultrastructural Alterations of Myelinated Fibers and Oligodendrocytes in the Prefrontal Cortex in Schizophrenia: A Postmortem Morphometric Study," Schizophrenia Research and Treatment 2011:325789 (2011); Uranova et al., "The Role of Oligodendrocyte Pathology in Schizophrenia," Int J Neuropsychopharmacol 10:537-545 (2007); Pruitt et al., "NCBI Reference Sequences (RefSeq): A Curated Non-Redundant Sequence Database of Genomes, Transcripts and Proteins," Nucleic Acids Research 35:D61-65 (2007)). 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 (Lee et al., "Oligodendroglia Metabolically Support Axons and Contribute to Neurodegeneration," Nature 487:443-448 (2012); Simons et al., "Oligodendrocytes: Myelination and Axonal Support," Cold Spring Harb Perspect Biol. (2015)). Yet despite genetic, cellular, pathological, and radiological studies that have correlated glial and myelin pathology with schizophrenia, the prevailing view is that that clinical hypomyelination among schizophrenics is secondary to neuronal pathology. Thus, the contribution of cell-autonomous glial dysfunction to schizophrenia has not been well studied, and consequently therapies targeting such dysfunctions have yet to be proposed.

[0004] The present disclosure is directed to overcoming these and other deficiencies in the art including with respect to:

[0005] Kolarik et al (1988) Act Nerv Super 30(2):155-157, which discusses therapeutic transplantation of human embryonic nerve tissue into a schizophrenic's brain.SUMMARY

[0006] The invention is as set out in the appended set of claims. In accordance with the claims, the invention relates to a preparation of glial progenitor cells for use in treating schizophrenia in a subject, wherein said preparation of glial progenitor cells is administered to a subject having schizophrenia at a dosage effective to treat schizophrenia in the subject. References herein to methods of treatment by administering the preparation containing glial progenitor cells in this description (including in its Examples) are to be interpreted as references to (pharmaceutical) preparations for use in a method for treatment of the human body by therapy (or for diagnosis).

[0007] Applicants have established that the contribution of cell autonomous glial dysfunction neurological disease can be investigated using a novel model of human glial chimeric mice (Windrem et al., "Neonatal Chimerization with Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008); Han et al., "Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning in Adult Mice," Cell Stem Cell 12:342-353 (2013); and Goldman et al., "Modeling Cognition and Disease Using Human Glial Chimeric Mice," Glia 63:1483-1493 (2015)) paired with the development of protocols for generating bipotential astrocyte-oligodendrocyte glial progenitor cells (GPCs) from patient-specific human induced pluripotent stem cells (hiPSCs) (Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013)). In these human glial chimeric mouse brains, the majority of resident glia are replaced by human glia and their progenitors (Windrem et al., "A Competitive Advantage by Neonatally Engrafted Human Glial Progenitors Yields Mice Whose Brains are Chimeric for Human Glia," The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 34:16153-16161 (2014)), allowing human glial physiology, gene expression, and effects on neurophysiological function to be assessed in vivo, in live adult mice (Han et al., "Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning in Adult Mice," Cell Stem Cell 12:342-353 (2013)). As described herein, the glial chimeric model was used to assess the contribution of human glia to schizophrenic disease phenotype. To this end, hGPCs were prepared from iPSCs derived from fibroblasts taken from either juvenile-onset schizophrenic (SCZ) patients or their normal controls. Differential gene expression of SCZ hGPCs was assessed relative to those of normal subjects, and the cells were transplanted into immunodeficient neonatal mice to produce patient-specific human glial chimeric mice. The glial chimeric mice were then analyzed in regard to the effects of SCZ derivation on astrocytic and oligodendrocytic differentiations in vivo, as well as on behavioral phenotype, and the data thereby obtained correlated to disease-associated gene expression. In accordance with the claimed invention, the applicants have identified novel therapeutic approaches for the treatment of schizophrenia.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 shows the functional and genomic assessment of schizophrenia-derived glial progenitor cells. This schematic summarizes the steps involved in the 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 3); in vivo astrocyte differentiation and phenotype (Figure 5); in vitro differential gene expression (Figure 6); and behavioral phenotype of the human glial chimeric host animals (Figure 10). Figures 2A-2H show CD140a+ glial progenitor cells are efficiently produced from both SCZ and normal hiPSCs. Flow cytometry for CD140a / PDGFaR+ glial progenitor cells (right plots, compared to unstained gating controls on left), reveals dominant proportions of CD140a-defined cells in both normal control patient-derived (top, Figures 2A-2B) and SCZ-derived (bottom, Figures 2C-2D) preparations. Figures 2A-2C and 2B-2D were run as matched pairs; Figures 2A and 2C show 177 and 168 days in vitro (DIV); Figures 2B and 2D show188 and 196 DIV. Figures 2E-2H show a representative post-FACS preparation of CD140a-sorted cells. Figure 2E is a phase image of cells immunostained for both olig2 (Figure 2F, red) and PDGFRα (Figures 2G; 2H, merged). These plots were typical of GPC cultures of both normal and SCZ-derived hiPSC lines. The sorted populations were used for genomics assessment, while both sorted and unsorted cells were used for transplantation, with no evident performance differences between the two. Figures 3A-3J show schizophrenia-derived hGPCs exhibit aberrant dispersal and relative hypomyelination. Human iPSC GPC chimeras were established by neonatal hGPC injection into shiverer mice. Chimeric mice were sacrificed at 19 weeks. GPCs derived from a control subject (Figure 3A) dispersed primarily in the major white matter tracts, whereas SCZ-derived GPCs (15 year old male) (Figure 3B) showed less white matter residence and more rapid cortical infiltration. Figures 3C-3D are sagittal sections that reveal callosal myelination by SCZ GPCs (Figure 3D) was less dense than that by control hGPCs (Figure 3C). Figures 3E-3F show higher power images from chimeric mice engrafted with hGPCs from 4 control patients (Figure 3E) vs. chimeric mice engrafted with hGPCs from 4 different SCZ patients (Figure 3F). Figure 3G shows 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). Figure 3H shows that 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 (Figure 3I) (p=0.0064, t-test) and transferrin (TFN) +< oligodendroglia (Figure 3J) (p<0.0001, t-test). Figures 4A-4B show schizophrenia-derived GPCs exhibit aberrant dispersal in vivo. The dispersal patterns of GPCs produced from SCZ patients typically differed from that of iPSC hGPCs derived from normal patients, in that SCZ GPCs did not remain and expand within the white matter before progressing to cortical infiltration, as was otherwise invariably the case with normal GPCs. Figures 4A-4B show 4 mice each implanted with either control subject-derived (line 22) or SCZ patient-derived (line 51) hGPCs. All SCZ hGPC-engrafted mice show disproportionate hGPC entry into the cortical and striatal gray matter, with less expansion and hence less net engraftment in the forebrain white matter tracts. This difference in hGPC dispersal pattern was noted consistently in all 4 SCZ lines assessed in vivo, each derived from a different patient, relative to their matched 4 control lines, similarly obtained from distinct patients (see Figure 3). Figure 5A-5J show astrocytic differentiation is impaired in schizophrenia hGPC chimeric brain. Human iPSC GPC chimeras were established in immunodeficient shiverer hosts and sacrificed at 19 weeks, for astrocytic differentiation assessment. Figures 5A-5B are representative images of the corpus callosum of mice neonatally injected with iPSC GPCs derived from either control (Figure 5A, line 22) or schizophrenic (Figure 5B, line 164) subjects (human nuclear antigen, green; glial fibrillary acidic protein, red). Figure 5A shows control hiPSC GPCs from all tested patients rapidly differentiated as GFAP+ astrocytes with dense fiber arrays in both callosal white and cortical gray matter. Figure 5B, in contrast, shows SCZ GPCs were slow to mature, with delayed GFAP expression. At 19 weeks, GFAP+ astrocyte densities were significantly greater in mice chimerized with control than SCZ-derived GPCs, both as a group (Figure 5C), and when analyzed line-by-line (Figure 5D). 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 (Figure 5E). Sholl analysis of individual astroglial morphologies (Sholl, D.A., "Dendritic Organization in the Neurons of the Visual and Motor Cortices of the Cat," J. Anat. 87:387-406 (1953)) as imaged in 150 µm sections and reconstructed in 3D by Neurolucida (Figure 5J), revealed that astrocytes in SCZ hGPC chimeras differed significantly from their control hGPC-derived counterparts, with fewer primary processes (Figure 5F), less proximal branching (Figure 5G), and longer distal fibers (Figure 5H). When the 3-D tracings (Figure 5J) were assessed by Fan-in radial analysis (MBF Biosciences) (Dang et al., "Formoterol, a Long-acting Beta2 Adrenergic Agonist, Improves Cognitive Function and Promotes Dendritic Complexity in a Mouse Model of Down Syndrome," Biol. Psychiatry 75:179-188 (2014)), control astrocytic processes were noted to extend uniformly in all directions, but SCZ astrocyte processes left empty spaces, indicative of a discontiguous domain structure (Figure 5I). ***p<0.0001, by t-test (Figures 5C, 5E, 5F, 5H; by 2-way ANOVA in Figure 5D; **p<0.002 in Figure 5I; p<0.0001 by non-linear comparison in Figure 5G. Scale, Figures 5A-5B = 50 µm, Figure 5J = 25 µm. Figures 6A-6G show schizophrenia-derived hGPCs suppress glial differentiation-associated gene expression. RNA sequence analysis reveals differential gene expression by SCZ hGPCs. Figure 6A shows an 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. Figure 6B is a network representation of functional annotations for the intersection gene list shown in Figure 6A. 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. Figure 6C shows top annotation terms identified for each module in Figure 6B. Figure 6D is a heatmap representation of 12 conserved differentially expressed genes that are associated to module 1 (grey in Figure 6B, 32.4%), which includes annotations related to neurotransmitter receptor and gated channel activity. Figure 6E is a heatmap representation of 15 conserved differentially expressed genes that are associated to module 2 (orange in Figure 6B, 28.7%), which comprises annotations related to cell-to-cell signaling and synaptic transmission. Figure 6F is a heatmap representation of 21 conserved differentially expressed genes that are associated to module 3 (dark blue in Figure 6B, 28.7%); annotations related to CNS and glial differentiation and development. Figure 6G is a heatmap representation of 4 conserved differentially expressed genes that are associated to module 4 (light blue in Figure 6B, 10.2%), with annotations related to myelination and lipid biosynthesis. The absolute expression in heatmaps is shown in UQ-normalized, log2-transformed counts (Li et al., "Comparing the Normalization Methods for the Differential Analysis of Illumina High-Throughput RNA-Seq Data," BMC Bioinformatics 16:347 (2015)). Figures 7A-7R show heat maps of significantly dysregulated genes in schizophrenic relative to control hiPSC GPCs. Expression patterns for shared genes differentially expressed by hiPSC GPCs derived from 4 schizophrenic patients, relative to the pooled gene expression pattern of hGPCs derived from 3 control-derived iPSCs (log2 fold change >1.0, FDR 5%, 118 genes total) are shown. The dysregulated genes were manually annotated and grouped into relevant sets based on their function and cellular localization. Each heat map visualizes UQ-normalized, log2-transformed counts of genes grouped into the following functional categories, comprising genes encoding: (Figure 7A) transcription regulators, zinc finger proteins, and other nucleus-associated proteins; (Figure 7B) glial differentiation-associated proteins; (Figure 7C) myelin-related genes and transcription factors; (Figure 7D) Wnt pathway effectors; (Figure 7E) metabolic enzymes; (Figure 7F) lipid and lipoprotein metabolism; (Figure 7G) kinases and phosphatases; (Figure 7H) adhesion molecules, cadherins, and astrotactins; (Figure 7I) GPCR signal intermediates; (Figure 7J) growth factors; (Figure 7K) cytokines; (Figure 7L) cell signaling and synaptic proteins; (Figure 7M) ion channels; (Figure 7N) transporters; (Figure 7O) extracellular matrix constituents; (Figure 7P) other transmembrane proteins; (Figure 7Q) other cytoplasmic and membrane-bound proteins; and (Figure 7R) unannotated genes, open reading frames, and long intergenic non-coding RNAs. Figure 8 shows expression of selected genes dysregulated in SCZ-derived GPCs as identified by RNA-seq analysis assessed by TaqMan Low Density Array (TLDA) RT-qPCR and compared against control GPCs. Expression data were normalized to GAPDH endogenous control. Mean ddCt values and standard error ranges calculated from 4 pooled SCZ GPC lines (n = 19) against 3 pooled control GPC lines (n = 10) are shown. The difference of expression in SCZ and control GPCs was assessed by paired t-test 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 proportion of undetermined and 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. Figure 9 shows neurexin-1 expression was suppressed in SCZ hGPCs. Western blots revealed that neurexin-1 protein was abundantly expressed by human GPCs purified by CD140a-directed FACS, and that neurexin-1 levels were lower in otherwise matched SCZ hGPCs (line 51 SCZ hGPCs vs. line 22 CTRL hGPCs). Figures 10A-10G show schizophrenia-derived human glial chimeras have significant behavioral abnormalities. Figures 10A-10E show behavioral tests that 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. Figure 10A shows prepulse inhibition studies. Normally-myelinated rag1- / - mice engrafted with SCZ hGPCs had reduced auditory pre-pulse inhibition (PPI) at all volumes of pre-pulse (Figure 10A). 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]). Figure 10B shows elevated plus maze studies. The left panel shows 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. The right panel shows 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). Figure 10C shows sucrose preference studies. 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). Figure 10D shows 3-chamber socialization test studies. 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 Figure 10D) 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 controls (left heat-map) (p=0.02; 3 SCZ lines, 34 mice; 3 control lines, 36 mice). Figure 10E shows novel object recognition studies. Mice engrafted with SCZ hGPCs showed significantly poorer novel object recognition (p=0.0006; 3 SCZ lines, 19 mice; 3 control lines, 28 mice). Figures 10F-10G demonstrate 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. Figure 10F shows the average distance traveled in meters / hr over a 72 hr period 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]. Figure 10G shows, on the left, sample heat-maps of one hour of activity during the light phase (16:00 hrs, 2nd 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. As shown on the 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. DETAILED DESCRIPTION

[0009] In accordance with the claimed invention, the present disclosure relates to a preparation of glial progenitor cells for use in the treatment of schizophrenia.

[0010] Schizophrenia is a chronic and severe mental disorder that affects how a person thinks, feels, and behaves. To date, there have been several suggested staging models of the disorder (Agius et al., "The Staging Model in Schizophrenia, and its Clinical Implications," Psychiatr. Danub. 22(2):211-220 (2010); McGorry et al., "Clinical Staging: a Heuristic Model and Practical Strategy for New Research and Better Health and Social Outcomes for Psychotic and Related Disorders," Can. J. Psychiatry 55(8):486-497 (2010); Fava and Kellner, "Staging: a Neglected Dimension in Psychiatric Classification," Acta Psychiatr. Scand. 87:225-230 (1993)). However, generally, schizophrenia develops in at least three stages: the prodromal phase, the first episode, and the chronic phase. There is also heterogeneity of individuals at all stages of the disorder, with some individuals considered ultra-high risk, clinical-high risk, or at-risk for the onset of psychosis (Fusar-Poli et al., "The Psychosis High-Risk State: a Comprehensive State-of-the-Art Review," JAMA Psychiatry 70:107-120 (2013)).

[0011] The preparation of glial progenitor cells for use in treating schizophrenia in a subject described herein is suitable for treating a subject in any stage of schizophrenia, and at any risk level of psychosis. For example, in one instance, a subject is a subject that is at risk for developing schizophrenia. Such a subject may have one or more genetic mutations in one or more genes selected from ABCA13, ATK1, C4A, COMT, DGCR2, DGCR8, DRD2, MIR137, NOS1AP, NRXN1, OLIG2, RTN4R, SYN2, TOP3B YWHAE, ZDHHC8, or chromosome 22 (22q11) that have been associated with the development of schizophrenia and may or may not be exhibiting any symptoms of the disease. In another instance, the subject may be in the prodromal phase of the disease and exhibiting one or more early symptoms of schizophrenia, such as anxiety, depression, sleep disorders, and / or brief intermittent psychotic syndrome. In another instance, the subject being treated with the preparation of glial progenitor cells described herein is experiencing psychotic symptoms, e.g., hallucinations, paranoid delusions, of schizophrenia.

[0012] As used herein, the term "subject" expressly includes human and non-human mammalian subjects. The term "non-human mammal" as used herein extends to, but is not restricted to, household pets and domesticated animals. Non-limiting examples of such animals include primates, cattle, sheep, ferrets, mice, rats, swine, camels, horses, poultry, fish, rabbits, goats, dogs and cats.

[0013] In accordance with aspects illustrated herein, the preparation of glial progenitor cells to be administered to the selected subject may be human or non-human. In one instance, the preparation of glial progenitor cells is a preparation of human glial progenitor cells.

[0014] Preferably the glial progenitor cells are bi-potential glial progenitor cells. In one instance, the glial progenitor cells are biased to producing oligodendrocytes. In another instance, the glial progenitor cells are biased to producing astrocytes.

[0015] Glial progenitor cells for use in the methods otherwise described herein can be derived from multipotent (e.g., neural stem cells) or pluripotent cells (e.g., embryonic stem cells or induced pluripotent stem cells) using methods known in the art or described herein.

[0016] In one instance, glial progenitor cells are derived from embryonic stem cells.

[0017] In one instance, glial progenitor cells are derived from induced pluripotential cells (iPSCs). "Induced pluripotent stem cells" as used herein refers to pluripotent cells that are derived from non-pluripotent cells, such as somatic cells or tissue stem cells. For example, and without limitation, iPSCs can be derived from, fetal, newborn, and adult tissue, from peripheral blood, umbilical cord blood, and bone marrow (see e.g., Cai et al., "Generation of Human Induced Pluripotent Stem Cells from Umbilical Cord Matrix and Amniotic Membrane Mesenchymal Cells," J. Biol. Chem. 285(15): 112227-11234 (2110); Giorgetti et al., "Generation of Induced Pluripotent Stem Cells from Human Cord Blood Cells with only Two Factors: Oct4 and Sox2," Nature Protocols, 5(4):811-820 (2010); Streckfuss-Bomeke et al., "Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts," Eur. Heart J. doi: 10.1093 / eurheartj / ehs203 (July 12, 2012); Hu et al., "Efficient Generation of Transgene-Free Induced Pluripotent Stem Cells from Normal and Neoplastic Bone Marrow and Cord Blood Mononuclear Cells," Blood doi: 10.1182 / blood-2010-07-298331 (Feb. 4, 2011); Sommer et al., "Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood using the STEMCCA Lentiviral Vector," J. Vis. Exp. 68: e4327 doi:10.3791 / 4327 (2012)). Exemplary somatic cells that can be used include fibroblasts, such as dermal fibroblasts obtained by a skin sample or biopsy, synoviocytes from synovial tissue, keratinocytes, mature B cells, mature T cells, pancreatic β cells, melanocytes, hepatocytes, foreskin cells, cheek cells, or lung fibroblasts (see e.g., Streckfuss-Bomeke et al., "Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts," Eur. Heart J. doi: 10.1093 / eurheartj / ehs203 (2012)). Although skin and cheek provide a readily available and easily attainable source of appropriate cells, virtually any cell can be used. Exemplary stem or progenitor cells that are suitable for iPSC production include, without limitation, myeloid progenitors, hematopoietic stem cells, adipose-derived stem cells, neural stem cells, and liver progenitor cells.

[0018] Autologous, allogenic, or xenogenic non-pluripotent cells can be used in order to produce the iPSCs used to generate the therapeutic glial progenitor cells. Allogenic cells for production of iPSCs, for example, are harvested from healthy donors (i.e., donors not having a neuropsychiatric disorder) and / or donor sources having suitable immunohistocompatibility. Xenogeneic cells can be harvested from a pig, monkey, or any other suitable mammal for the production if iPSCs. Autologous non-pluripotent cells can also be harvested from the same subject to be treated. However, such autologous cells require genetic manipulation and / or other treatment prior to therapeutic administration. In particular, as described herein expression of a number of genes (see Table 2) are dysregulated in neuropsychiatric disorders. Accordingly, autologous cells are preferably genetically modified and / or otherwise treated to correct the dysregulation so that they exhibit normal, non-disease related expression and / or activity levels prior to administration.

[0019] Induced pluripotent stem cells can be produced by expressing a combination of reprogramming factors in a somatic cell. Suitable reprogramming factors that promote and induce iPSC generation include one or more of Oct4, Klf4, Sox2, c-Myc, Nanog, C / EBPα, Esrrb, Lin28, and Nr5a2. In some instances, at least two reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In other instances, at least three reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In other instances, at least four reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell.

[0020] iPSCs may be derived by methods known in the art including the use of integrating viral vectors (e.g., lentiviral vectors, inducible lentiviral vectors, and retroviral vectors), excisable vectors (e.g., transposon and floxed lentiviral vectors), and non-integrating vectors (e.g., adenoviral and plasmid vectors) to deliver the aforementioned genes that promote cell reprogramming (see e.g., Takahashi and Yamanaka, Cell 126:663-676 (2006); Okita. et al., Nature 448:313-317 (2007); Nakagawa et al., Nat. Biotechnol. 26:101-106 (2007); Takahashi et al., Cell 131:1-12 (2007); Meissner et al. Nat. Biotech. 25:1177-1181 (2007); Yu et al. Science 318:1917-1920 (2007); Park et al. Nature 451:141-146 (2008); and U.S. Patent Application Publication No. 2008 / 0233610). Other methods for generating IPS cells include those disclosed in WO2007 / 069666, WO2009 / 006930, WO2009 / 006997, WO2009 / 007852, WO2008 / 118820, U.S. Patent Application Publication Nos. 2011 / 0200568 to Ikeda et al., 2010 / 0156778 to Egusa et al., 2012 / 0276070 to Musick, and 2012 / 0276636 to Nakagawa, Shi et al., Cell Stem Cell 3(5): 568-574 (2008), Kim et al., Nature 454: 646-650 (2008), Kim et al., Cell 136(3) :411-419 (2009), Huangfu et al., Nature Biotechnology 26: 1269-1275 (2008), Zhao et al., Cell Stem Cell 3: 475-479 (2008), Feng et al., Nature Cell Biology 11: 197-203 (2009), and Hanna et al., Cell 133(2): 250-264 (2008).

[0021] Integration free approaches, i.e., those using non-integrating and excisable vectors, for deriving iPSCs free of transgenic sequences are particularly suitable in the therapeutic context. Suitable methods of iPSC production that utilize non-integrating vectors include methods that use adenoviral vectors (Stadtfeld et al., "Induced Pluripotent Stem Cells Generated without Viral Integration," Science 322: 945-949 (2008), and Okita et al., "Generation of Mouse Induced Pluripotent Stem Cells without Viral Vectors," Science 322: 949-953 (2008)), Sendi virus vectors (Fusaki et al., "Efficient Induction of Transgene-Free Human Pluripotent Stem Cells Using a Vector Based on Sendi Virus, an RNA Virus That Does Not Integrate into the Host Genome," Proc Jpn Acad. 85: 348-362 (2009)), polycistronic minicircle vectors (Jia et al., "A Nonviral Minicircle Vector for Deriving Hyman iPS Cells," Nat. Methods 7: 197-199 (2010)), and self-replicating selectable episomes (Yu et al., "Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences," Science 324: 797-801 (2009)). Suitable methods for iPSC generation using excisable vectors are described by Kaji et al., "Virus-Free Induction of Pluripotency and Subsequent Excision of Reprogramming Factors," Nature 458: 771-775 (2009), Soldner et al., "Parkinson's Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors," Cell 136:964-977 (2009), Woltjen et al., "PiggyBac Transposition Reprograms Fibroblasts to Induced Pluripotent Stem Cells," Nature 458: 766-770 (2009), and Yusa et al., "Generation of Transgene-Free Induced Pluripotent Mouse Stem Cells by the PiggyBac Transposon," Nat. Methods 6: 363-369 (2009). Suitable methods for iPSC generation also include methods involving the direct delivery of reprogramming factors as recombinant proteins (Zhou et al., "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins," Cell Stem Cell 4: 381-384 (2009)) or as whole-cell extracts isolated from ESCs (Cho et al., "Induction of Pluripotent Stem Cells from Adult Somatic Cells by Protein-Based Reprogramming without Genetic Manipulation," Blood 116: 386-395 (2010)).

[0022] The methods of iPSC generation described above can be modified to include small molecules that enhance reprogramming efficiency or even substitute for a reprogramming factor. These small molecules include, without limitation, epigenetic modulators such as the DNA methyltransferase inhibitor 5'-azacytidine, the histone deacetylase inhibitor VPA, and the G9a histone methyltransferase inhibitor BIX-01294 together with BayK8644, an L-type calcium channel agonist. Other small molecule reprogramming factors include those that target signal transduction pathways, such as TGF-β inhibitors and kinase inhibitors (e.g., kenpaullone) (see review by Sommer and Mostoslavsky, "Experimental Approaches for the Generation of Induced Pluripotent Stem Cells," Stem Cell Res. Ther. 1:26 doi:10.1186 / scrt26 (2010)).

[0023] Suitable iPSCs derived from adult fibroblasts can be obtained following the procedure described in Streckfuss-Bomeke et al., "Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts," Eur. Heart J. doi: 10.1093 / eurheartj / ehs203 (2012)). iPSCs derived from umbilical cord blood cells can be obtained as described in Cai et al., "Generation of Human Induced Pluripotent Stem Cells from Umbilical Cord Matrix and Amniotic Membrane Mesenchymal Cells," J. Biol. Chem. 285(15): 112227-11234 (2110) and Giorgetti et al., "Generation of Induced Pluripotent Stem Cells from Human Cord Blood Cells with only Two Factors: Oct4 and Sox2," Nature Protocols, 5(4):811-820 (2010). iPSCs derived from bone marrow cells can be obtained using methods described in Streckfuss-Bomeke et al., "Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts," Eur. Heart J. doi: 10.1093 / eurheartj / ehs203 (July 12, 2012), and Hu et al., "Efficient Generation of Transgene-Free Induced Pluripotent Stem Cells from Normal and Neoplastic Bone Marrow and Cord Blood Mononuclear Cells," Blood doi: 10.1182 / blood-2010-07-298331 (Feb. 4, 2011)). iPSCs derived from peripheral blood can be obtained following the methods described in Sommer et al., "Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood using the STEMCCA Lentiviral Vector," J. Vis. Exp. 68: e4327 doi:10.3791 / 4327 (2012). iPS cells contemplated for use in the methods described herein are not limited to those described in the above references, but rather include cells prepared by any method as long as the cells have been artificially induced from cells other than pluripotent stem cells.

[0024] Methods of obtaining highly enriched preparations of oligodendrocyte progenitor cells from the iPSCs or embryonic stem cells (e.g., human embryonic stem cells) that are suitable for the claimed invention are disclosed in WO2014 / 124087 to Goldman and Wang, and Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitors Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12(2):252-264 (2013).

[0025] Briefly, oligodendrocyte progenitor cells are derived from a pluripotent population of cells, i.e., iPSCs or embryonic stem cells, using a protocol that directs the pluripotent cells through serial stages of neural and glial progenitor cell differentiation. Each stage of lineage restriction is characterized and identified by the expression of certain cell proteins. Stage 1 of this process involves culturing the pluripotent cell population under conditions effective to induce embryoid body formation. As described herein, the pluripotent cell population may be maintained in co-culture with other cells, such as embryonic fibroblasts, in an embryonic stem cell (ESC) media (e.g., DMEM / F12 containing a suitable serum replacement and bFGF). The pluripotent cells are passaged before reaching 100% confluence, e.g., 80% confluence, when colonies are approximately 250-300µm in diameter. The pluripotential state of the cells is readily assessed using markers to SSEA4, TRA-1-60, OCT-4, NANOG, and / or SOX2.

[0026] To generate embryoid bodies (EBs) (Stage 2), which are complex three-dimensional cell aggregates of pluripotent stem cells, pluripotent cell cultures are dissociated once they achieved ~80% confluence with colony diameters at or around 250-300µm. The EBs are initially cultured in suspension in ESC media without bFGF, and then switched to neural induction medium supplemented with bFGF and heparin. To induce neuroepithelial differentiation (Stage 3) EBs are plated and cultured in neural induction medium supplemented with bFGF, heparin, laminin, then switched to neural induction media supplemented with retinoic acid. Neuroepithelial differentiation is assessed by the co-expression of PAX6 and SOX1, which characterize central neural stem and progenitor cells.

[0027] To induce pre-oligodendrocyte progenitor cell ("pre-OPCs") differentiation, neuroepithelial cell colonies are cultured in the presence of additional factors including retinoic acid, B27 supplement, and a sonic hedgehog (shh) agonist (e.g., purmophamine). The appearance of pre-OPC colonies is assessed by the presence of OLIG2 and / or NKX2.2 expression. While both OLIG2 and NKX2.2 are expressed by central oligodendrocyte progenitor cells, NKX2.2 is a more specific indicator of oligodendroglial differentiation. Accordingly, an early pre-oligodendrocyte progenitor cell stage is marked by OLIG +< / NKX2.2 -< cell colonies. OLIG +< / NKX2.2 -< early pre-OPCs are differentiated into later-stage OLIG +< / NKX2.2 +< pre-OPCs by replacing retinoic acid with bFGF. At the end of Stage 5, a significant percentage of the cells are pre-OPCs as indicated by OLIG2 +< / NKX2.2 +< expression profile.

[0028] Pre-OPCs are further differentiated into bipotential oligodendrocyte progenitor cells by culture in glial induction media supplemented with growth factors such as triiodothyronine (T3), neurotrophin 3 (NT3), insulin growth factor (IGF-1), and platelet-derived growth factor-AA (PDGF-AA) (Stage 6). These culture conditions can be extended for 3-4 months or longer to maximize the production of myelinogenic oligodendrocyte progenitor cells when desired. Cell preparations suitable for transplantation into an appropriate subject are identified as containing PDGFRα +< oligodendrocyte progenitor cells.

[0029] Alternative methods of obtaining preparations of oligodendrocyte progenitor cells from the iPSCs or embryonic stem cells that are known in the art can also be used to produce a therapeutic population of cells for use in the claimed invention. In yet another instance, glial progenitor cells can be extracted from adult brain tissue containing a mixed population of cells directly by using the promoter specific separation technique, as described in U.S. Patent Application Publication Nos. 20040029269 and 20030223972 to Goldman. In that instance, the glial progenitor cells are isolated from ventricular or subventricular zones of the brain or from the subcortical white matter.

[0030] In some instances, it may be preferable to enrich a cell preparation comprising oligodendrocyte progenitor cells to increase the concentration and / or purity of the therapeutic oligodendrocyte progenitor cells prior to administration. Accordingly, in one instance, the A2B5 monoclonal antibody (mAb) that recognizes and binds to gangliosides present on glial progenitor cells early in the developmental or differentiation process can be used to separate glial progenitor cells from a mixed population of cells (Nunes et al., "Identification and Isolation of Multipotential Neural Progenitor Cells From the Subcortical White Matter of the Adult Human Brain.," Nat Med. 9(4):439-47 (2003)). Using the A2B5 mAb, glial progenitor cells can be separated, enriched, or purified from a mixed population of cell types. In another instance, selection of CD140α / PDGFRα positive cells is employed to produce a purified or enriched preparation of bipotential glial progenitor cells. In another instance, selection of CD9 positive cells is employed to produce a purified or enriched preparation of oligodendrocyte-biased progenitor cells. In yet another instance, both CD140α / PDGFRα and CD9 positive cell selection is employed to produce a purified or enriched preparation of oligodendrocyte progenitor cells. In a further instance, selection of CD44 positive cells is employed to produce a purified or enriched preparation of astrocyte-biased progenitor cells (Liu et al., "CD44 Expression Identifies Astrocyte-Restricted Precursor Cells," Dev. Biol. 276(1):31-46 (2004)). In another instance, both CD140α / PDGFRα and CD44 positive cell selection is employed to produce a purified or enriched preparation of oligodendrocyte progenitor cells. In another instance, CD140α / PDGFRα, CD9, and CD44 positive cell selection is employed to produce a purified or enriched preparation of oligodendrocyte progenitor cells.

[0031] The glial progenitor cell preparation to be administered is optionally negative for a PSA-NCAM marker and / or other neuronal lineage markers, and / or negative for one or more inflammatory cell markers, e.g., negative for a CD11 marker, negative for a CD32 marker, and / or negative for a CD36 marker (which are markers for microglia). Optionally, the preparation of glial progenitor cells is negative for any combination or subset of these additional markers. Thus, for example, the preparation of glial progenitor cells is negative for any one, two, three, or four of these additional markers.

[0032] The selected preparation of administered glial progenitor cells may comprise at least 80% glial progenitor cells, including, for example, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% glial progenitor cells. The selected preparation of glial progenitor cells can be relatively devoid (e.g., containing less than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of other cells types such as neurons or cells of neuronal lineage, fibrous astrocytes and cells of fibrous astrocyte lineage, multipotent cells, and pluripotential stem cells (like ES cells). Optionally, exemplary cell populations are substantially pure populations of glial progenitor cells.

[0033] Positive and / or negative selection for cell markers of interest (e.g., PDGFRα marker, A2B5 marker, and / or a CD44 marker) can be carried out serially or sequentially and can be performed using conventional methods known in the art such as immunopanning. The selection methods optionally involve the use of fluorescence sorting (FACS), magnetic sorting (MACS), or any other method that allows rapid, efficient cell sorting. Examples of methods for cell sorting are taught for example in U.S. Patent No. 6,692,957.

[0034] Generally, cell sorting methods use a detectable moiety. Detectable moieties include any suitable direct or indirect label, including, but not limited to, enzymes, fluorophores, biotin, chromophores, radioisotopes, colored beads, electrochemical, chemical-modifying or chemiluminescent moieties. Common fluorescent moieties include fluorescein, cyanine dyes, coumarins, phycoerythrin, phycobiliproteins, dansyl chloride, Texas Red, and lanthanide complexes or derivatives thereof.

[0035] One of skill in the art readily appreciates how to select for or against a specific marker. Thus, by way of example, a population of cells sorted for a particular marker includes identifying cells that are positive for that particular marker and retaining those cells for further use or further selection steps. A population of cells sorted against a specific marker includes identifying cells that are positive for that particular marker and excluding those cells for further use or further selection steps.

[0036] The glial progenitor cell preparations for use in the claimed invention, including the enriched preparations, can be optionally expanded in culture to increase the total number of cells for therapeutic administration. The cells can be expanded by either continuous or pulsatile exposure to PDGF-AA or AB as mitogens that support the expansion of oligodendrocyte progenitor cells; they can be exposed to fibroblast growth factors, including FGF2, FGF4 , FGF8 and FGF9, which can support the mitotic expansion of the glial progenitor cells, but which can bias their differentiation to a mixed population of astrocytes as well as oligodendrocytes. The cells can also be expanded in media supplemented with combinations of FGF2, PDGF, and NT3, which can optionally be supplemented with either platelet-depleted or whole serum (see Nunes et al. "Identification and Isolation of Multipotent Neural Progenitor Cells from the Subcortical White Matter of the Adult Human Brain," Nature Medicine 9:239-247; Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nature Medicine 10:93-97 (2004)).

[0037] The glial progenitor cell population may be administered bilaterally into multiple sites of the subject being treated as described in Han et al., "Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning Adult Mice," Cell Stem Cell 12:342-353 (2013) and Wang et al., "Human iPSCs-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12: 252-264 (2013)). Methods for transplanting nerve tissues and cells into host brains are described by Bjorklund and Stenevi (eds), Neural Grafting in the Mammalian CNS, Ch. 3-8, Elsevier, Amsterdam (1985); U.S. Patent No. 5,082,670 to Gage et al.; and U.S. Patent No. 6,497,872 to Weiss et al.. Typical procedures include intracerebral, intraventricular, intrathecal, and intracisternal administration.

[0038] The glial progenitor cell preparation can be delivered directly to the forebrain subcortex, specifically into the anterior and posterior anlagen of the corpus callosum. Glial progenitor cells can also be delivered to the cerebellar peduncle white matter to gain access to the major cerebellar and brainstem tracts. Glial progenitor cells can also be delivered to the spinal cord.

[0039] Alternatively, the cells may be placed in a ventricle, e.g. a cerebral ventricle. Grafting cells in the ventricle may be accomplished by injection of the donor cells or by growing the cells in a substrate such as 30% collagen to form a plug of solid tissue which may then be implanted into the ventricle to prevent dislocation of the graft cells. For subdural grafting, the cells may be injected around the surface of the brain after making a slit in the dura.

[0040] Delivery of the cells to the subject can include either a single step or a multiple step injection directly into the nervous system. Although adult and fetal oligodendrocyte precursor cells disperse widely within a transplant recipient's brain, for widespread neuropsychiatric disorders, multiple injections sites can be performed to optimize treatment. Injection is optionally directed into areas of the central nervous system such as white matter tracts like the corpus callosum (e.g., into the anterior and posterior anlagen), dorsal columns, cerebellar peduncles, cerebral peduncles. Such injections can be made unilaterally or bilaterally using precise localization methods such as stereotaxic surgery, optionally with accompanying imaging methods (e.g., high resolution MRI imaging). One of skill in the art recognizes that brain regions vary across species; however, one of skill in the art also recognizes comparable brain regions across mammalian species.

[0041] In one instance, the oligodendrocyte progenitor cell preparation is injected as dissociated cells. In another instance, the oligodendrocyte progenitor cell preparation is provided as non-dissociated cells. In either case, the cellular transplants optionally comprise an acceptable solution. Such acceptable solutions include solutions that avoid undesirable biological activities and contamination. Suitable solutions include an appropriate amount of a pharmaceutically-acceptable salt to render the formulation isotonic. Examples of the pharmaceutically-acceptable solutions include, but are not limited to, saline, Hank's Balanced Salt Solution, Ringer's solution, dextrose solution, and culture media. The pH of the solution is preferably from 5 to 8, and more preferably from 7 to 7.5.

[0042] The injection of the dissociated cellular transplant can be a streaming injection made across the entry path, the exit path, or both the entry and exit paths of the injection device. Suitable injection devices include cannula, needle, insertion tube, cannula guided by an insertion tube. Automation and stereotactic positioning systems can be used to provide precise delivery to targeted regions with a uniform entry and exit speed and an injection speed and volume.

[0043] The number of glial progenitor cells administered to the subject can range from 10 2< -10 9< at each administration (e.g., injection site), depending on the size and species of the recipient, and the volume of tissue requiring cell replacement. Single administration (e.g., injection) doses can span ranges of 1 x 10 3< - 9 x 10 3< , 1 x 10 4< - 9 x 10 4< , 1 x 10 5< - 9 x 10 5< , 1 x 10 6< - 9 x 10 6< , 1 x 10 7< - 9 x 10 7< , 1 x 10 8< - 9 x 10 8< , 1 x 10 9< - 9 x 10 9< , 1 x 10 3< - 9 x 10 3< , or any amount in total for a transplant recipient patient. In one instance, the administered dose is 1x 10 7< - 4 x 10 7< cells. To achieve such doses, cell preparation having a concentration of 1-2 x 10 3< cells / µl, 1-2 x 10 4< cells / µl, 1-2 x 10 5< cells / µl, 1-2 x 10 6< cells / µl, 1-2 x 10 7< cells / µl of pharmaceutically acceptable carrier are prepared. In one instance, the cell preparation for administration has a concentration of 1 x 10 5< - 2 x 10 5< cells / µl in a total volume of 25 µl to 50 µl.

[0044] Since the CNS is an immunologically privileged site, administered cells, including xenogeneic, can survive and, optionally, no immunosuppressant drugs or a typical regimen of immunosuppressant agents are used in the treatment methods. However, optionally, an immunosuppressant agent may also be administered to the subject prior to and after receiving the cell therapy. Immunosuppressant agents and their dosing regimens are known to one of skill in the art and include such agents as Azathioprine, Azathioprine Sodium, Cyclosporine, Daltroban, Gusperimus Trihydrochloride, Sirolimus, Mycophenolate mofetil (MMF) and Tacrolimus. In one instance, a combination of any of the aforementioned immunosuppressant agents are administered to the subject. In one instance, a combination of MMF and tacrolimus are administered to the subject. Dosages ranges and duration of the regimen can be varied with the disorder being treated; the extent of rejection; the activity of the specific immunosuppressant employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the specific immunosuppressant employed; the duration and frequency of the treatment; and drugs used in combination. One of skill in the art can determine acceptable dosages for and duration of immunosuppression. The dosage regimen can be adjusted by the individual physician in the event of any contraindications or change in the subject's status.

[0045] In one instance, one or more immunosuppressant agents are administered to the subject starting at 10 weeks prior to cell administration. In one instance, the one or more immunosuppressant agents are administered to the subject starting at 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, < 24 hours prior to cell administration. In one instance, one or more immunosuppressant agents are administered to the subject starting on the day of cell administration and continuing for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months post administration. In one instance, the one or more immunosuppressant agents are administered to the subject for > 1 year following administration.

[0046] As used herein, "treating" or "treatment" refers to any indication of success in amelioration of an injury, pathology, or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neurological examination, and / or psychiatric evaluation. In accordance with the claimed invention, "treating" refers to the administration of glial progenitor cells to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms of schizophrenia. "Therapeutic effect" refers to the reduction, elimination, or prevention of schizophrenia or symptoms of schizophrenia in the subject. Treatment may be prophylactic (to prevent or delay the onset or worsening of schizophrenia, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of schizophrenia.

[0047] A "dosage effective to treat," as used herein refers to the amount of cells that is effective for production of a desired result. This amount varies, for example, depending upon the health and physical condition of the individual to be treated, the mental and emotional capacity of the individual, the degree of protection desired, the formulation, the attending physician's assessment of the medical situation, and other relevant factors.EXAMPLES

[0048] The examples below are intended to exemplify the practice of instances of the disclosure but are by no means intended to limit the scope of the claimed invention.Materials and Methods for Examples

[0049] Patient identification, protection and sampling. Patients from which these lines were derived were diagnosed with disabling degrees of schizophrenia with onset in early adolescence; all patients and their guardians were consented by a child psychiatrist (RLF) under an approved protocol of Case Western School of Medicine, blinded as to subsequent line designations, and no study investigators had access to patient identifiers.

[0050] iPSC line derivation and production of GPCs. Punch biopsies of the skin were obtained from patients with juvenile onset schizophrenia (ages 10 to 17 years old) and controls (ages 24 to 32 years old). Induced pluripotent stem cells (iPSC) lines were derived from the patient samples using an excisable floxed polycistronic hSTEMCCA lentiviral vector. Short tandem repeat (STR)-based DNA fingerprinting was used to confirm iPSC identity, as a match to original patient or control donor. Additional genotyping was performed using Illumina Omni5 SNP arrays. The iPSCs were then driven toward a glial progenitor cell (GPC) fate using previously described protocols (Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitor Cells can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013)). Cells were harvested between 160-240 DIV, by which time most typically expressed the bipotential GPC marker PDGFαR / CD140a, while the remainder were A2B5+ / CD140a- astrocytes. The karyotypes of all iPSC lines were assessed during glial differentiation to ensure genotypic stability of the cells utilized in all experiments presented here (karyotyping by WiCell, Madison, WI). All iPSCs showed a normal karyotype, except for line 51, which was found to have a balanced Robertsonian translocation of chromosome 13, an anomaly previously associated with juvenile-onset schizophrenia (Graw et al., "Isochromosome 13 in a Patient with Childhood-Onset Schizophrenia, ADHD, and Motor Tic Disorder," Mol Cytogenet 5:2 (2012)).

[0051] Host transplantation. Homozygous shiverer mice (The Jackson Laboratory, Bar Harbor, ME) were crossed with homozygous rag2-null immunodeficient mice (Shinkai et al., "RAG2-Deficient Mice Lack Mature Lymphocytes Owing to Inability to Initiate V(D)J Rearrangement," Cell 68:855-867 (1992)) on the C3h background (Taconic, Germantown, NY, USA) to generate shi / shi x rag2 - / -< myelin-deficient, immunodeficient mice (Windrem et al., "Neonatal Chimerization with Human Glial Progenitor Cells can both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008)). In addition, rag1 - / -< normally-myelinated immunodeficient mice (B6.129S7-Rag1 tm1Mom< / J), were obtained from the Jackson Laboratory and bred in the lab. Suspensions of single-cells or small clusters of hiPSC-derived GPCs were spun down to 100,000 cells / µl. Neonates were anesthetized by cooling, and transplanted bilaterally in the corpus callosum with a total of 200,000 cells, as described (Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat.Med. 10:93-97 (2004)). At 3 months of age (shi / shi x rag2 - / -< ) or after completion of behavioral testing at 6-9 months (rag1 - / -< only), transplanted mice were anesthetized with pentobarbital, then perfusion fixed with cold HBSS + / +< followed by 4% paraformaldehyde (PF) with a 2 hour post-fixation in cold PF. All procedures were approved by the University of Rochester's Committee on Animal Resources.

[0052] Immunolabeling. Brains were cryopreserved, embedded in OCT (Tissue-Tek OCT, Sakura Finetek, Torrance, CA) and sectioned at 20 µm, either sagittally or coronally, on a cryostat. Human cells were identified with mouse antihuman nuclei, clone 235-1 at 1:800 (MAB1281, EMD Millipore, Billerica, MA). Myelin basic protein was labeled with rat anti-MBP at 1:25 (Ab7349, Abcam, Cambridge, MA), oligodendrocyte progenitors with anti-human-specific PDGF Receptor α (D13C6, XP ®< rabbit mAb 5241, 1:300, Cell Signaling Technology), oligodendrocytes with mouse anti-human-specific transferrin (clone HT1 / 13.6.3, 08691231, MP Biomedicals), astrocytes with anti-human-specific GFAP (SMI 21 at 1:1000, Covance, Princeton, NJ). Alexa Fluor secondary antibodies, goat anti- mouse, rat, and rabbit 488, 568, 594, and 647 were used at 1:400 (Life Technologies, Carlsbad, CA).Antibodies and dilutions used

[0053] Antigen Name Dilution Catalog Company hGFAPMouse anti-human (specific) GFAP1:500SMI-21RCovancehNAMouse anti-human nuclear antigen, cl. 235-11:800MAB1281MilliporehNG2Mouse anti-NG2, clone 9.2.271:200MAB2029MilliporeMBPRat anti-Myelin Basic Protein1:25ab7349AbcamOlig2Rabbit anti-Olig21:500RA25017NeuromicsPDGFRαRabbit anti-PDGFRα, clone D13C61:3005241SCell SignlTransferrinMouse anti-human transferrin1:800ab9538AbcamSecondary antibodiesAlexaFluor 568 Goat anti-Mouse IgG (H+L)1:400A-11031InvitrogenAlexaFluor 568 Goat anti-Mouse IgG11:400A-21124InvitrogenAlexaFluor 488 Goat anti-Mouse IgG (H+L)1:400A-11029InvitrogenAlexaFluor 488 Goat anti-Mouse IgG11:400A-21121InvitrogenAlexaFluor 568 Goat anti-Rabbit IgG (H+L)1:400A-11036InvitrogenAlexaFluor 488 Goat anti-Rabbit IgG (H+L)1:400A-11034InvitrogenCy5 Goat anti-Rat1:400A10525InvitrogenAlexaFluor 568 Goat anti-Rat IgG (H+L)1:400A-11077InvitrogenAlexaFluor 488 Goat anti-Rat IgG (H+L)1:400A-11006Invitrogen

[0054] Western blots. GPCs derived from CWRU22 and CWRU51 were sorted by FACS for CD140a at DIV160-200, directly into cell lysis buffer (NP40, Invitrogen, FNN0021) with protease inhibitor (Roche, 183617025) on ice. The insoluble fraction was removed by centrifugation at 12,000 g for 5 minutes at 4°C, and the supernatant analyzed for total protein with BCATM Protein Assay Kit (Thermo, 23227). 10 µg sample aliquots were separated on 4-12% gradient gels by SDS-PAGE electrophoresis (XCell SureLock, Invitrogen, 071210). Separated protein was transferred to PVDF membranes, which were blocked with 5% dry milk and incubated sequentially with a rabbit polyclonal anti-neurexin-1 antisera (Millipore, ABN161-1, 1:1000) at 4°C overnight, then washed and followed serially by a mouse monoclonal anti-β actin (Abcam, ab173838, 1:5000) at RT for 1h, and anti-mouse and anti-rabbit secondary antibodies (GE Healthcare, 95107-322 and 95107-328, 1:10000) at RT for 1h. Membranes were visualized by chemiluminescence (Mix ECLTM Reagent, GE Healthcare, RPN2236) through exposure of X-ray film. Experiments were repeated 3 times, with 3 different sets of cells.

[0055] Imaging and Quantitative histology. For mapping the distribution of human nuclei, or photographing gross distribution of myelin at low power, whole brain sections were imaged on a Leica LMD 6500. Imaging for phenotypic counts was performed on an Olympus BX51 driven by Stereo Investigator software (MBF, Williston, VT).

[0056] Astrocyte morphometrics. Shiverer x rag2-null mice were sacrificed at 4.5 months of age and their white matter astrocyte morphologies assessed. 150 µm thick coronal slices were taken by Vibratome at Bregma -1.0 mm from control (22, 37 and C27) or SCZ (51, 164, 193) hGPC-engrafted mice, incubated in mouse anti-hGFAP for 1 week, then 4 hrs in Alexa 568 goat anti-mouse. The slices were mounted on slides and imaged at 100x by confocal (Leica SP8). The images were traced using Neurolucida 360 (MicroBrightfield, Inc.). Individual astrocytes were selected from the middle of the corpus callosum at mid-depth so as to capture cells and their processes in their entirety. Nine cells / brain were analyzed by Neurolucida with Sholl analysis, as 3 cells / slice and 3 slices / brain, taken at 500, 1000, and 1500 µm lateral of the midline. Two or three brains were assessed for each of three lines produced from separate patients, for a total of 8 brains and 72 traced cells / condition, for both CTRL and SCZ-engrafted groups. For Sholl analysis, concentric shells placed at successively increasing diameters of 5 µm were centered on the cell body, and the number of intersections between cell processes and shells counted (Sholl, DA., "Dendritic Organization in the Neurons of the Visual and Motor Cortices of the Cat," J. Anat. 87:387-406 (1953)). For the assessment and quantitative description of astrocytic fiber 3D architecture, Fan-in analysis (MBF Biosciences) was used as previously described for studies of dendritic topology (Dang et al., "Formoterol, a Long-Acting Beta2 Adrenergic Agonist, Improves Cognitive Function and Promotes Dendritic Complexity in a Mouse Model of Down Syndrome," Biol. Psychiatry 75:179-188 (2014) ).

[0057] Myelin luminance analysis. To measure forebrain myelination, luminance analysis based on measurement of myelin basic protein (MBP) immunofluorescence. Evenly-spaced and uniformly sampled coronal sections were stained for MBP as described, and images taken at 10x using a Nikon Ni-E and Nikon DS-Fi1 camera. The corpus callosum was selected as region of interest, and mean intensity values were obtained using NIS Elements v.4.5.

[0058] Behavior. Behavioral tests were scored using either ANY-maze (Stoelting, Wood Dale, IL) or EthoVision (Noldus). Behavioral testing began at either 25 weeks (for pre-pulse inhibition) or 30-36 weeks (all other tests) and typically lasted 3 weeks; starting age was matched between experimentals and controls. A total of 6-12 recipient mice were engrafted and tested per cell line, or 17-36 mice per group for each behavioral comparison, with a roughly equal balance of male (M) and female (F) recipients. Tests were performed in the same sequence for all mice and included: 1) Elevated Plus Maze. Each test mouse was placed in the center of a raised, plus-shaped apparatus, consisting of 2 enclosed arms and 2 open arms, facing an open arm (Walf et al., "The Use of the Elevated Plus Maze as an Assay of Anxiety-Related Behavior in Rodents," Nat Protoc 2:322-328 (2007)). Each tested mouse was videotaped and scored for time spent in the open vs. closed arms. 2) Three chamber social choice. The test apparatus is a plexiglass enclosure divided into thirds with connecting doors (Ugo Basile, Italy) (Yang et al., "Automated Three-Chambered Social Approach Task for Mice," Curr Protoc Neurosci, Chapter 8, Unit 8, 26 (2011)). Each test mouse was first acclimated to the central chamber or 5 minutes. The doors to the outer chambers were then removed, and the test mouse allowed to explore all three chambers for 10 minutes. The test mouse was then guided back to the central chamber, and a same sex and age stranger mouse was placed in a cylindrical container in one side chamber, while an empty cylindrical container was placed in the opposite side chamber. The mouse was then recorded for 10 minutes and scored with respect to the amount of time it spent with the stranger mouse vs. the empty compartment. 3) Novel Object Recognition. Each test mouse was placed in an empty 1 ft 2< testing chamber for 5 minutes to acclimate, then removed, and two identical objects were placed in the chamber. The mouse was returned to the chamber with the objects, placed facing directly away from them, recorded for 10 minutes and scored for time spent in proximity to each object (Bevins et al., "Object Recognition in Rats and Mice: A One-Trial Non-Matching-to-Sample Learning Task to Study 'Recognition Memory'," Nat Protoc 1:1306-1311 (2006)). After one hour, the experiment was repeated, with one of the two objects replaced by a novel object. 4) Pre-pulse inhibition. Each mouse was placed in a restraint chamber inside a larger isolation cabinet, equipped with sound, light, and air puff generators (SR-LAB, San Diego Instruments), and auditory PPI assessed as described (Geyer et al., "Measurement of Startle Response, Prepulse Inhibition, and Habituation," Curr Protoc Neurosci, Chapter 8, Unit 8, 7 (2001)). 5) Sucrose preference. This experiment was always performed last, as mice were individually housed in order to measure liquid consumption. Sucrose preference was determined by the percentage of sucrose water consumed as a proportion of all water consumed (Willner et al., "Reduction of Sucrose Preference by Chronic Unpredictable Mild Stress, and its Restoration by a Tricyclic Antidepressant," Psychopharmacology (Berl) 93:358-364 (1987)). Water is delivered in the colony by Hydropac (Lab Products, Inc.), so an additional Hydropac containing sucrose water was added to the cage and the two packs were weighed daily.

[0059] Activity and sleep assessment. Individually-housed mice were video recorded in 12" x 12" x 13.5" acrylic chambers, using infra-red cameras during the dark phase, for 72 continuous hours under 12 / 12 light / dark conditions. The distance traveled in meters per hour was calculated by Noldus Ethovision software, and averaged across 8 CTRL mice (gray fill, lines 22 and 17) and 10 SCZ mice (purple fill, line 52). In addition, transitions between phases of the light cycle (measured 30 minutes before and 30 minutes after light changes) were analyzed in terms of the number of consecutive seconds of immobility as a percentage of total immobility (AnyMaze, Stoelting), per 30 min measurement block as described (McShane et al., "Characterization of the Bout Durations of Sleep and Wakefulness," J. Neurosci. Methods 193:321-333 (2010); Pack et al., "Novel Method for High-Throughput Phenotyping of Sleep in Mice," Physiol. Genomics 28:232-238 (2007)).

[0060] Statistical analysis. Unless otherwise noted, analyses were done in GraphPad Prism v.7. Individual tests were performed as noted for each experiment. All data are presented as mean ± SEMs.

[0061] RNA-seq and bioinformatics. hGPCs assessed for gene expression were first sorted by fluorescence-activated cell sorting on the basis of the cell surface marker CD140a (BD Pharmingen) as described (Figure 3) (Sim et al., "CD140a Identifies a Population of Highly Myelinogenic, Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nature Biotechnology 29:934-941 (2011)). Using polyA-selection, mRNA was isolated from these PDGFRα+ hGPCs, which were derived from iPSCs made from 4 patients with juvenile-onset schizophrenia (SCZ line numbers 8 [n=4 independent cell preparations], 29 [n=3], 51 [n=7], and 164 [n=8]); and 3 demographically similar healthy controls (CTR lines 22 [n=3], 37 [n=4], and 205 [n=7]). Sequencing libraries were prepared using the TruSeq RNA v2 kit, and sequenced on an Illumina HiSeq 2500 platform for approximately 45 million 1x100 bp reads per sample. The sequencing reads were pre-processed by trimming off adapter and low-quality sequences from the 3' end using Trimmomatic (Bolger et al., "Trimmomatic: A Flexible Trimmer for Illumina Sequence Data," Bioinformatics 30:2114-2120 (2014)). The quality of reads before and after pre-processing was assessed with FastQC (D'Antonio et al., "RAP: RNA-Seq Analysis Pipeline, A New Cloud-Based NGS Web Application," BMC Genomics 16:S3 (2015)), and the pre-processed reads were then aligned to the RefSeq NCBI reference human genome version GRCh38 (Pruitt et al., "NCBI Reference Sequences (RefSeq): A Curated Non-Redundant Sequence Database of Genomes, Transcripts and Proteins," Nucleic Acids Research 35:D61-65 (2007)) with Subread read aligner (Liao et al., "The Subread Aligner: Fast, Accurate and Scalable Read Mapping by Seed-and-Vote," Nucleic Acids Research 41:e108 (2013)) using Hamming distance to break ties between more than one optimal mapping locations. Raw gene counts were obtained from BAM alignment files with the featureCounts tool (Liao et al., "Feature Counts: An Efficient General Purpose Program for Assigning Sequence Reads to Genomic Features," Bioinformatics 30:923-930 (2014)). After eliminating lowly expressed transcripts with a count <5 reads in more than 5 samples across the dataset, the count data was normalized using the RUVSeq (Risso et al., "Normalization of RNA-Seq Data Using Factor Analysis of Control Genes or Samples," Nat Biotechnol 32:896-902 (2014)) R Bioconductor package (Gentleman et al., "Bioconductor: Open Software Development for Computational Biology and Bioinformatics," Genome Biology 5:R80 (2004)) to account for variance. As described in the RUVSeq manual, the normalization was accomplished in the following three-step procedure: 1) in silico negative control genes were determined by first-pass differential expression analysis by the edgeR (Robinson et al., "EdgeR: A Bioconductor Package for Differential Expression Analysis of Digital Gene Expression Data," Bioinformatics 26:139-140 (2010)) and DESeq2 (Love et al., "Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2," Genome Biology 15:550 (2014)) R Bioconductor packages, taking genes with FDR-adjusted P values >0.75, as calculated by both methods (approximately 7000 genes were unaffected by the condition of interest); 2) the negative control genes were then used in the RUVs function of the RUVSeq package, for calculation of variance factors; and, 3) the second-pass differential expression analysis (5% FDR and log2 fold change >1) for determining disease-dysregulated genes was performed using the original counts, adjusting for RUVs-calculated variance factors by multi-factor GLM models implemented in the edgeR and DESeq2 packages.

[0062] This three-step analysis, with filtering for low-expressed transcripts, was used to compare each SCZ-derived hGPC cell line to the pooled CTR-derived hGPCs. The intersection of the resulting 4 individual lists of differentially expressed genes was taken as the conserved representative list of SCZ-dysregulated genes. In the normalization procedure for each comparison, the number of RUVs-calculated variance factors was limited to 1 for line 29, 3 for lines 8 and 164, and 7 for line 51, as determined by principal component and hierarchical clustering analyses performed with native R functions. To obtain average fold changes and P values for dysregulated genes in all 4 SCZ hGPC lines, a differential expression comparison of pooled SCZ to pooled CTR lines was performed by the same filtering and analysis workflow with the number of variance factors limited to 9.

[0063] For all differential expression comparisons, only the significant results that agreed between edgeR and DESeq2 methods were used in downstream analysis. Once individual fold changes and P values for dysregulated genes in all 4 SCZ hGPC lines were established relative to control lines, the differential expression of pooled SCZ to pooled CTR lines was performed. For each SCZ cell line, separate DE comparisons were performed against each control line and the intersection of the DE genes was taken as a representative list for that SCZ line against the control population. Fold changes and FDR-adjusted P values reported were calculated by edgeR. Functional annotation of the conserved set of SCZ-dysregulated genes was done using ToppCluster (Kaimal et al., "ToppCluster: A Multiple Gene List Feature Analyzer for Comparative Enrichment Clustering and Network-Based Dissection of Biological Systems," Nucleic Acids Research 38:W96-102 (2010)) and Ingenuity Pathway Analysis (IPA). Network visualization and analysis of the results of functional annotation were performed in Gephi (Jacomy et al., "ForceAtlas2, A Continuous Graph Layout Algorithm for Handy Network Visualization Designed for the Gephi Software," PloS one 9:e98679 (2014)) graph visualization software.

[0064] For streamlined execution of the above data processing and analysis routines, a set of Python and R scripts was developed. All genomic data have been deposited to GEO, accession number GSE86906.

[0065] Real-Time PCR. Expression levels in SCZ- and control derived GPCs of selected target genes identified by RNA-seq were assayed by TaqMan Low Density Array (TLDA) Real-Time PCR. The raw data were analyzed in EspressionSuite Software version 1.1 supplied by Applied Biosystems and exported into HTqPCR R package (Chambers et al., "Highly Efficient Neural Conversion of Human ES and iPS cells by Dual Inhibition of SMAD Signaling," Nat Biotechnol 27:275-280 (2009)) for relative quantification analysis.Example 1 - Generation of iPSCs from Patients with Juvenile-Onset Schizophrenia

[0066] Patients with juvenile-onset schizophrenia, 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 then isolated from each sample; from these, 11 new independent hiPS cell lines were derived from 8 patient samples (5 juvenile-onset schizophrenia patients and 3 healthy gender-matched and age-analogous controls (Table 1). Table 1 Subject number hiPSC Line(s) Age of subject Gender Ethnicity RNA-Seq of CD140a +< GPCs Anatomic assessment N=shiverer mice Behavioral assessment N=myelin w / t mice Control Subjects Cntrl 1 19, 22 26MC√√√Cntrl 2 37 32FAA√√√Cntrl 3 205 25MC√√√Cntrl 4 C27 NANANA√√Schizophrenic Subjects SCZ 1 8 10FC√√SCZ 2 51, 52 16MC√√√SCZ 3 29, 31 12MC√√SCZ 4 164 14FAA√√SCZ 5 193 15FNA√√ Patients and cell lines used in this study. A total of 11 new independent iPS cell lines were derived from 8 subjects; 5 juvenile-onset schizophrenic patients and 3 healthy controls; an established control line (C27) from an additional normal subject was published previously (Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitor Cells can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013); Chambers et al., "Highly Efficient Neural Conversion of Human ES and iPS cElls by Dual Inhibition of SMAD Signaling," Nat Biotechnol 27:275-280 (2009)). hGPCs derived from these cells were assigned to individual experiments as noted. C, Caucasian; AA, African-American; NA, not available.

[0067] iPSC were generated using excisable floxed polycistronic hSTEMCCA lentivirus (Zou et al., "Establishment of Transgene-Free Induced Pluripotent Stem Cells Reprogrammed from Human Stem Cells of Apical Papilla for Neural Differentiation," Stem Cell Res Ther 3:43 (2012); Somers et al., "Generation of Transgene-Free Lung Disease-Specific Human Induced Pluripotent Stem Cells Using a Single Excisable Lentiviral Stem Cell Cassette," Stem Cells 28:1728-1740 (2010)) encoding Oct4, Sox2, Klf4 and c-Myc (Takahashi et al., "Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors," Cell 131:861-872 (2007); Welstead et al., "Generating iPS Cells from MEFS Through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4," J Vis Exp (2008)). 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 (Chambers et al., "Highly Efficient Neural Conversion of Human ES and iPS Cells by Dual Inhibition of SMAD Signaling," Nat Biotechnol 27:275-280 (2009)), was also used, to ensure that the control engraftment and differentiation data were consistent with prior studies (Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013)). Altogether, hGPC preparations were evaluated from 7 iPSC lines derived from 5 SCZ patients, and 5 iPSC lines derived from 4 control subjects (Table 1). The iPSC cells were then instructed to GPC fate as previously described (Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013)), 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 (Figure 2) (Sim et al., "CD140a Identifies a Population of Highly Myelinogenic, Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nature Biotechnology 29:934-941 (2011)). To optimize glial differentiation in vivo, transplants were limited to those preparations in which most cells were CD140a+ GPCs, with the remainder astroglial.

[0068] It was first asked whether SCZ hGPCs differed from wild-type hGPCs in myelination competence. To this end, SCZ hGPCs were implanted into neonatal immunodeficient shiverer mice (rag2 - / -< x MBP shi / shi< ), a congenitally hypomyelinated mutant lacking myelin basic protein (MBP) (Rosenbluth, J., "Central Myelin in the Mouse Mutant Shiverer," J Comp Neural 194:639-648 (1980); Roach et al., "Characterization of Cloned cDNA Representing Rat Myelin Basic Protein: Absence of Expression in Brain of Shiverer Mutant Mice," Cell 34:799-806 (1983)). 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 (Windrem et al., "Neonatal Chimerization with Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008); Windrem et al., "A Competitive Advantage by Neonatally Engrafted Human Glial Progenitors Yields Mice Whose Brains are Chimeric for Human Glia," The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 34:16153-16161 (2014)). By this means, mice with patient-specific, largely humanized forebrain white matter, derived from SCZ or control subjects were established (Figures 3A-3D).Example 2 - SCZ Glial Chimeric Mice Were Uniformly Hypomyelinated

[0069] It was 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 3A), as was previously noted in both fetal tissue- and hiPSC GPC-engrafted shiverer mice (Windrem et al., "Neonatal Chimerization with Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008); Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013)). 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 vs. paired controls) (Figure 3B and Figure 4). This resulted in significantly fewer donor hGPCs in the white matter of shiverers engrafted with SCZ GPCs (Figures 3H-3I and Figure 4). Importantly, this was associated with substantially diminished central myelination in these mice, as reflected by both MBP immunostaining (Figures 3C-3D and 3E-3F) and myelin luminance (Figure 3G).

[0070] Since the SCZ hGPC-engrafted shiverers manifested deficient myelination, it was asked whether this was due to a relative failure of SCZ hGPCs to remain within white matter, or rather to a cell-intrinsic failure in myelinogenesis. Examining 19 wk-old SCZ and control hGPC-engrafted shiverer mice, significantly fewer human nuclear antigen (hNA)-defined donor-derived cells were found in SCZ hGPC-engrafted shiverer white matter (40,615 ± 2,189 x 10 3< hNA +< cells / mm3 ,< n=18) than in mice identically transplanted with control hGPCs (69,970 ± 4,091 / mm 3< ; n=32; p<0.0001 by 2-tailed t test (Fagerland et al., "Performance of five Two-Sample Location Tests For Skewed Distributions with Unequal Variances," Contemp Clin Trials 30:490-496 (2009); Merman, D. W., "A Note on Preliminary Tests of Equality of Variances," Br J Math Stat Psychol 57:173-181 (2004)) (Figure 3H). Moreover, the numbers of hNA+ donor cells co-expressing the oligodendroglial lineage marker Olig2 were similarly depressed in the SCZ hGPC-engrafted mice (33,619 ± 2,435 / mm 3< , n=26), relative to control hGPC-engrafted mice (46,139 ± 2,858 / mm 3< , n=17; p<0.002) (Figure 3I). On that basis, it was 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 3< , n=25; vs. 17,754±2,023 / mm 3< , n=17, respectively; p=0.0006, Mann-Whitney) (Figure 3J). 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.Example 3 - SCZ Glial Chimeric Mice Manifested Developmentally-Delayed Astrocytic Maturation

[0071] It was 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) at 19 weeks after neonatal graft, using a species-specific anti-human GFAP antibody. It was 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 5A-5B). 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, such that whereas all control hGPC forebrains showed dense human GFAP +< astrocytic maturation, far fewer SCZ hGPCs manifested hGFAP expression and astrocytic phenotype (controls: 6,616 ± 672.3 GFAP +< cells / mm 3< in callosum, n=12; SCZ: 1,177 ± 276.6 GFAP +< callosal cells / mm 3< , n=19; p<0.0001 by 2-way t-test (Figure 5C). This defect in astrocytic differentiation was consistently observed in all mice (n=19) derived from the 3 SCZ patients assessed, compared to the control GPC-engrafted mice (n=12) derived from 3 normal subjects (Figure 5D), and reflected in part the lower proportion of GFAP +< astrocytes that developed among engrafted human cells in the SCZ HGPC-engrafted brains (Figure 5E). Furthermore, Sholl analysis of individual astroglial morphologies (Sholl, D.A., "Dendritic Organization in the Neurons of the Visual and Motor Cortices of the Cat," J. Anat. 87:387-406 (1953)), as imaged in 150 µm sections and reconstructed in Neurolucida (Figure 5J), revealed that astrocytes in SCZ hGPC chimeras differed significantly from their control hGPC-derived counterparts, with fewer primary processes (Figure 5F), less proximal branching (Figure 5G), longer distal fibers (Figure 5H), and less coherent domain structure (Figure 5I). Thus, SCZ hGPCs derived from multiple patients exhibited a common defect in phenotypic maturation, and hence proved deficient in astrocytic differentiation as well as myelination.Example 4 - SCZ hGPCs Showed Cell-Autonomous Misexpression of Differentiation-Associated Genes

[0072] 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, RNA-seq analysis was used to identify the differentially expressed genes of SCZ iPSC-derived GPCs, relative to those of control-derived glia. Sequencing data was used to reconstruct the transcriptional patterns of hGPCs derived from 4 different SCZ and 3 control patients. hGPCs were derived at time points ranging from 154 to 242 days in vitro and sorted for hGPCs using CD140a-targeted FACS. Using a 5% FDR and a fold-change threshold of 2, a total of 118 mRNAs were identified that were differentially expressed by CD140a-sorted SCZ hGPCs relative to their control iPSC hGPCs (Figures 6A-6B). Among those 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 down-regulated in the SCZ hGPCs relative to their normal controls (Figures 6C and 6F). 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 6G; see Table 2 and Figure 7 for detailed gene expression data). Table 2 Significantly dysregulated genes in SCZ-derived relative to control-derived OPCs (list 1 of 5) Gene IDLog2 FCP ValueEntrez Gene NameTranscription regulators, & nucleus-associated (10 genes) SLFN132.8322.18E-13schlafen family member 13SLFN112.1936.47E-09schlafen family member 11NLRP2-7.2084.24E-58NLR family, pyrin domain containing 2SOX10-4.4488.37E-19SRY-box 10NR0B1-3.3215.92E-27nuclear receptor subfamily 0 group B member 1OLIG2-3.1964.23E-19oligodendrocyte lineage transcription factor 2OLIG1-3.1465.72E-22oligodendrocyte transcription factor 1ZNF439-2.1396.14E-08zinc finger protein 439IRX1-1.8155.52E-06iroquois homeobox 1ZNF488-1.4644.44E-07zinc finger protein 488Glial differentiation (5 genes) SOX10-4.4488.37E-19SRY-box 10OLIG2-3.1964.23E-19oligodendrocyte lineage transcription factor 2OLIG1-3.1465.72E-22oligodendrocyte transcription factor 1DLL3-2.3522.09E-24delta-like 3 (Drosophila)MPZ-2.1057.66E-13myelin protein zeroMyelination-associated (12 genes) SOX10-4.4488.37E-19SRY-box 10GPR17-3.3571.94E-10G protein-coupled receptor 17UGT8-3.2504.36E-09UDP glycosyltransferase 8OLIG2-3.1964.23E-19oligodendrocyte lineage transcription factor 2GAL3ST1-2.6814.01E-12galactose-3-O-sulfotransferase 1CNTN1-2.6755.65E-15contactin 1PLLP-2.5811.21E-24plasmolipinOMG-2.5615.09E-12oligodendrocyte myelin glycoproteinFA2H-2.4404.45E-08fatty acid 2-hydroxylaseSLC8A3-2.2242.00E-12solute carrier family 8 (sodium / calcium exchanger), member 3MPZ-2.1057.66E-13myelin protein zeroZNF488-1.4644.44E-07zinc finger protein 488Wnt signaling (4 genes) WNT7B-2.6261.46E-07wingless-type MMTV integration site family member 7BPCDH15-2.5301.42E-10protocadherin-related 15PCDH11X-2.3641.25E-08protocadherin 11 X-linkedCDH10-1.6469.33E-07cadherin 10Significantly dysregulated genes in SCZ-derived relative to control-derived OPCs (list 2 of 5) Gene IDLog2 FCP ValueEntrez Gene NameEnzymes (15 genes) SLFN132.8322.18E-13schlafen family member 13SLFN112.1936.47E-09schlafen family member 11HS3ST42.1491.44E-04heparan sulfate-glucosamine 3-sulfotransferase 4ALOX51.7772.35E-05arachidonate 5-lipoxygenaseCA10-3.5503.07E-08carbonic anhydrase XNEU4-3.3618.31E-40neuraminidase 4 (sialidase)UGT8-3.2504.36E-09UDP glycosyltransferase 8GAL3ST1-2.6814.01E-12galactose-3-O-sulfotransferase 1CNTN1-2.6755.65E-15contactin 1CSMD3-2.5605.78E-10CUB and Sushi multiple domains 3GALNT13-2.4672.80E-11polypeptide N-acetylgalactosaminyltransferase 13FA2H-2.4404.45E-08fatty acid 2-hydroxylaseAOAH-2.2712.99E-12acyloxyacyl hydrolaseKIF19-1.6445.10E-06kinesin family member 19DSEL-1.1511.35E-10dermatan sulfate epimerase-likeLipid & lipoprotein metabolism (5 genes) ALOX51.7772.35E-05arachidonate 5-lipoxygenaseNEU4-3.3618.31E-40neuraminidase 4 (sialidase)GAL3ST1-2.6814.01E-12galactose-3-O-sulfotransferase 1PLPPR1-2.6521.16E-09phospholipid phosphatase related 1PLPPR5-1.5731.69E-09phospholipid phosphatase related 5Kinases & phosphatases (6 genes) PTPRT4.4101.69E-17protein tyrosine phosphatase, receptor type TPPP1R16B-2.6651.29E-09protein phosphatase 1 regulatory subunit 16BPPAPDC1A-2.3576.76E-09phospholipid phosphatase 4DGKG-2.3069.29E-12diacylglycerol kinase gammaEPHB1-1.3443.89E-17EPH receptor B1PNCK-1.2264.25E-08pregnancy up-regulated nonubiquitous CaM kinaseAdhesion molecules (5 genes) DSCAM-3.1481.58E-12Down syndrome cell adhesion moleculeASTN2-2.2424.70E-21astrotactin 2OPCML-2.0997.87E-10opioid binding protein / cell adhesion molecule-likeBAI1-2.0001.31E-12adhesion G protein-coupled receptor B1CDH10-1.6469.33E-07cadherin 10GPCR signaling (10 genes) CCL21.8321.97E-07chemokine (C-C motif) ligand 2GPR17-3.3571.94E-10G protein-coupled receptor 17GPR45-2.8952.95E-14G protein-coupled receptor 45WNT7B-2.6261.46E-07wingless-type MMTV integration site family member 7BGPR139-2.5891.32E-08G protein-coupled receptor 139OMG-2.5615.09E-12oligodendrocyte myelin glycoproteinCRHR1-2.3825.81E-12corticotropin releasing hormone receptor 1DGKG-2.3069.29E-12diacylglycerol kinase gammaBAI1-2.0001.31E-12adhesion G protein-coupled receptor B1GPR123-1.8071.76E-18adhesion G protein-coupled receptor A1Significantly dysregulated genes in SCZ-derived relative to control-derived OPCs (list 3 of 5) Gene IDLog2 FCP ValueEntrez Gene NameGrowth factors (3 genes) FGF14-2.0215.14E-07fibroblast growth factor 14FGF12-1.9885.47E-09fibroblast growth factor 12CSPG5-1.2856.38E-17chondroitin sulfate proteoglycan 5Cytokines (2 genes) CCL21.8321.97E-07chemokine (C-C motif) ligand 2CMTM5-3.0233.69E-15CKLF-like MARVEL transmembrane domain containing 5Synaptic-junction associated (32 genes) CCL21.8321.97E-07chemokine (C-C motif) ligand 2ALOX51.7772.35E-05arachidonate 5-lipoxygenaseBRIN P3-3.4334.70E-22bone morphogenetic protein / retinoic acid inducible neural-specific 3DSCAM-3.1481.58E-12Down syndrome cell adhesion moleculeKCND2-3.1455.67E-11potassium channel, voltage gated Shal related subfamily D, member 2NXPH1-2.8923.11E-15neurexophilin 1CHRNA4-2.7554.45E-14cholinergic receptor, nicotinic alpha 4ARHGAP36-2.6861.21E-05Rho GTPase activating protein 36CNTN1-2.6755.65E-15contactin 1NETO1-2.6332.52E-12neuropilin and tolloid like 1PLLP-2.5811.21E-24plasmolipinPCDH15-2.5301.42E-10protocadherin-related 15GRIA4-2.5191.84E-09glutamate receptor, ionotropic, AMPA 4BCAN-2.4737.35E-32brevicanGABRA3-2.4501.08E-12gamma-aminobutyric acid (GABA) A receptor, alpha 3CRHR1-2.3825.81E-12corticotropin releasing hormone receptor 1SHISA7-2.3021.43E-14shisa family member 7SLC8A3-2.2242.00E-12solute carrier family 8 (sodium / calcium exchanger), member 3MPZ-2.1057.66E-13myelin protein zeroGRID2-2.0553.65E-06glutamate receptor, ionotropic, delta 2RPH3A-2.0141.01E-06rabphilin 3AVWC2-2.0039.67E-13von Willebrand factor C domain containing 2CTTN BP2-1.9047.21E-08cortactin binding protein 2MT3-1.7955.27E-09metallothionein 3KCNA3-1.7193.03E-12potassium channel, voltage gated shaker related subfamily A, member 3BRINP2-1.6252.03E-12bone morphogenetic protein / retinoic acid inducible neural-specific 2LGI3-1.6144.07E-08leucine-rich repeat LGI family member 3SLC6A1-1.5965.52E-11solute carrier family 6 (neurotransmitter transporter), member 1GRID1-1.5627.61E-07glutamate receptor, ionotropic, delta 1GRIK4-1.5472.73E-07glutamate receptor, ionotropic, kainate 4KCNK9-1.4604.40E-06potassium channel, two pore domain subfamily K, member 9EPHB1-1.3443.89E-17EPH receptor B1Ion channels (10 genes) KCND2-3.1455.67E-11potassium channel, voltage gated Shal related subfamily D, member 2GRIA4-2.5191.84E-09glutamate receptor, ionotropic, AMPA 4GABRA3-2.4501.08E-12gamma-amino butyric acid (GABA) A receptor, alpha 3ASIC4-2.3214.13E-14acid sensing ion channel subunit family member 4KCNJ9-2.1872.85E-21potassium channel, inwardly rectifying subfamily J, member 9GRID2-2.0553.65E-06glutamate receptor, ionotropic, delta 2KCNA3-1.7193.03E-12potassium channel, voltage gated shaker related subfamily A, member 3GRID1-1.5627.61E-07glutamate receptor, ionotropic, delta 1GRIK4-1.5472.73E-07glutamate receptor, ionotropic, kainate 4KCNK9-1.4604.40E-06potassium channel, two pore domain subfamily K, member 9Significantly dysregulated genes in SCZ-derived relative to control-derived OPCs (list 4 of 5) Gene IDLog2 FCP ValueEntrez Gene NameTransporters (6 genes) AZGP1-3.3231.66E-08alpha-2-glycoprotein 1, zinc-bindingPLLP-2.5811.21E-24plasmolipinSNX22-2.2973.53E-20sorting nexin 22SLC8A3-2.2242.00E-12solute carrier family 8 (sodium / calcium exchanger), member 3RPH3A-2.0141.01E-06rabphilin 3ASLC6A1-1.5965.52E-11solute carrier family 6 (neurotransmitter transporter), member 1Extracellular matrix-associated (14 genes) CCL21.8321.97E-07chemokine (C-C motif) ligand 2COL20A1-3.4767.33E-14collagen, type XX, alpha 1BRINP3-3.4334.70E-22bone morphogenetic protein / retinoic acid inducible neural-specific 3WNT7B-2.6261.46E-07wingless-type MMTV integration site family member 7BBCAN-2.4737.35E-32brevicanFGF14-2.0215.14E-07fibroblast growth factor 14VWC2-2.0039.67E-13von Willebrand factor C domain containing 2FGF12-1.9885.47E-09fibroblast growth factor 12LUZP2-1.9487.22E-06leucine zipper protein 2ELFN2-1.9431.47E-06extracellular Leu-rich repeat and fibronectin type III domain containing 2KY-1.8732.41E-13kyphoscoliosis peptidaseBRINP2-1.6252.03E-12bone morphogenetic protein / retinoic acid inducible neural-specific 2LGI3-1.6144.07E-08leucine-rich repeat LGI family member 3CSPG5-1.2856.38E-17chondroitin sulfate proteoglycan 5Transmembrane proteins (5 genes) TMEM257-4.0185.58E-23transmembrane protein 257CMTM5-3.0233.69E-15CKLF-like MARVEL transmembrane domain containing 5CHRNA4-2.7554.45E-14cholinergic receptor, nicotinic alpha 4LINGO1-2.1393.56E-14leucine-rich repeat and Ig domain containing 1TMEM229B-1.1877.76E-06transmembrane protein 229BCytoplasmic & membrane-associated (15 genes) CCL21.8321.97E-07chemokine (C-C motif) ligand 2BTN3A21.3558.10E-20butyrophilin subfamily 3 member A2BEX5-3.8852.55E-21brain expressed X-linked 5ARHGAP36-2.6861.21E-05Rho GTPase activating protein 36WNT7B-2.6261.46E-07wingless-type MMTV integration site family member 7BGRIA4-2.5191.84E-09glutamate receptor, ionotropic, AMPA 4SNX22-2.2973.53E-20sorting nexin 22LHFPL3-2.2501.71E-07lipoma HMGIC fusion partner-like 3RPH3A-2.0141.01E-06rabphilin 3ACTTNBP2-1.9047.21E-08cortactin binding protein 2SHC3-1.8307.88E-19SHC (Src homology 2 domain containing) transforming protein 3MT3-1.7955.27E-09metallothionein 3LGI3-1.6144.07E-08leucine-rich repeat LGI family member 3KCNK9-1.4604.40E-06potassium channel, two pore domain subfamily K, member 9CSPG5-1.2856.38E-17chondroitin sulfate proteoglycan 5Significantly dysregulated genes in SCZ-derived relative to control-derived OPCs (list 5 of 5) Gene IDLog2 FCP ValueEntrez Gene NamelincRNAs, ORFs, & uncharacterized (25 genes) RPS12P111.6479.02E-30not availableLOC101927206-3.5922.74E-63not availableLOC100505797-3.2663.10E-27myosin heavy chain IB-likeC10orf85-3.0813.52E-10long intergenic non-protein coding RNA 1561CXXC11-2.8147.70E-19receptor (chemosensory) transporter protein 5 (putative)LOC284395-2.7921.20E-10uncharacterized LOC284395RP11-547I7.2-2.7794.81E-34not availableAC009227.2-2.7583.05E-13not availableLOC101928430-2.6915.56E-10not availableFLJ38379-2.3891.25E-15not availableLOC102723927-2.3637.74E-21uncharacterized LOC102723927RP11-1055B8.3-2.3361.02E-16not availableAC131097.3-2.1805.80E-16not availableClorf94-2.0777.03E-11chromosome 1 open reading frame 94HMGA1P7-2.0574.49E-09high mobility group AT-hook 1 pseudogene 7LOC101929249-2.0031.52E-06uncharacterized LOC101929249LOC101927646-2.0011.22E-10uncharacterized LOC101927646AC010890.1-1.9373.53E-10not availableC2orf80-1.9246.74E-18chromosome 2 open reading frame 80MAPT-AS1-1.8445.40E-07MAPT antisense RNA 1LOC100128127-1.8141.43E-09not availableRP11-1134I14.8-1.7835.72E-22not availableSLC6A1-AS1-1.7061.31E-09SLC6A1 antisense RNA 1KIAA1244-1.6431.63E-10ARFGEF family member 3FAM222A-AS1-1.2971.10E-11FAM222A antisense RNA 1

[0073] Table 2. Significantly dysregulated genes in SCZ relative to control GPCs. These tables list shared genes differentially expressed by hiPSC GPCs derived from 4 schizophrenic patients, relative to the pooled gene expression pattern of hGPCs derived from 3 control-derived iPSCs (log2 fold change >1.0, FDR 5%, 116 genes total, red, upregulated in SCZ vs CTRL; green, downregulated in SCZ GPCs; color intensity proportionate to differential dysregulation). The fold- changes (FC) and FDR-adjusted p values shown here were derived from the comparison of the pooled schizophrenia-derived GPC cell lines to the pooled control-derived GPC lines. The dysregulated genes were grouped into functional sets according to their cellular roles and localizations.

[0074] These expression data suggest that the diminished myelination of SCZ hGPC-transplanted shiverer brains reflected aberrant oligodendrocytic differentiation from the engrafted SCZ hGPCs. Similarly, since 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 for 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 schizophrenia. 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 6D-6E). These disease-linked channel and synapse-associated genes were largely down-regulated in the SCZ hGPCs, and included a number of potassium channel genes (Figure 6D), including KCND2, KCNJ9, KCNK9 and KCNA3, as well as a number of transcripts associated with synaptic development and function (Figure 6E and Table 2). The latter included NXPH1, NLGN3, and LINGO 1, among others (Table 3), synaptic genes whose dysregulation has been previously linked to both SCZ and the autism spectrum disorders (Sudhof, T. C., "Neuroligins and Neurexins Link Synaptic Function to Cognitive Disease," Nature 455:903-911 (2008); Andrews et al., "A Decade From Discovery to Therapy: Lingo-1, the Dark Horse in Neurological and Psychiatric Disorders," Neurosci Biobehav Rev 56:97-114 (2015); Fernandez-Enright et al., "Novel Implications of Lingo-1 and its Signaling Partners in Schizophrenia," Translational psychiatry 4:e348 (2014); Mackowiak et al., "Neuroligins, Synapse Balance and Neuropsychiatric Disorders," Pharmacol Rep 66:830-835 (2014); Salyakina et al., "Copy Number Variants in Extended Autism Spectrum Disorder Families Reveal Candidates Potentially Involved in Autism Risk," PloS one 6:e26049 (2011)). Table 3 SCZ.08 vs. Pooled CTRSCZ.29 vs. Pooled CTRSCZ.51 vs. Pooled CTRSCZ.164 vs. Pooled CTRSCZ.08+29+51+16 vs. Pooled CTRGene IDLog2 FCP ValueLog2 FCP ValueLog2 FCP ValueLog2 FCP ValueLog2 FCP ValueDSCAML1 -1.9718.83E-07-2.9681.31E-04-1.0898.32E-05NSNS-0.9825.34E-0LINGO1 -2.3203.29E-04-2.8852.26E-04-1.4523.36E-06-2.5239.19E-12-2.1393.56E-1NLGN1 -1.2493.37E-04-1.0144.32E-02NSNS-0.5453.88E-02-0.6253.15E-0NLGN2 NSNS-0.7671.74E-02-0.3843.01E-03-0.4081.47E-02-0.4523.37E-0NLGN3 -0.5633.98E-02-1.6693.07E-03-1.0111.63E-09-0.9583.67E-05-1.1431.61E-1NRP1 -1.3621.15E-031.4095.00E-04NSNSNSNSNSNSNRP2 NSNS1.5482.07E-03NSNS-0.7333.65E-02NSNSNRXN1 -3.2595.93E-06-2.9845.55E-03-1.1761.35E-02NSNS-1.1612.04E-0NRXN2 NSNS-2.1799.04E-06-1.2522.71E-11-0.7203.33E-03-1.1026.82E-1NRXN3 1.8741.60E-04NSNSNSNS1.1981.20E-020.9096.89E-0NTNG2 -0.8747.99E-03-1.8143.51E-04NSNSNSNSNSNSNXPE3 NSNSNSNSNSNS0.4101.74E-020.2482.79E-0NXPH1 -3.0193.98E-07-3.6661.84E-03-1.3381.06E-02-2.3172.46E-09-2.8923.11E-1NXPH2 -2.2885.62E-04NSNSNSNSNSNSNSNSNXPH3 NSNS-2.2251.49E-03-1.0847.92E-03NSNS-0.6752.66E-0NXPH4 -2.1865.96E-034.0954.08E-14NSNS-1.5601.09E-02NSNSPTPRZ1 NSNS-2.9672.60E-05-0.7924.13E-03-0.8705.38E-03-1.2962.41E-1RGS4 -1.9826.80E-052.1843.97E-041.1057.04E-04-1.4192.64E-03NSNSSLITRK2 -6.8121.35E-04NSNS-7.3077.51E-25-9.3215.54E-06-6.1385.52E-1SLITRK3 -2.9582.68E-03NSNS-1.6986.37E-04-3.4901.61E-11-2.5026.76E-0SLITRK4 -4.1577.12E-05NSNS-3.7135.02E-05-2.6781.20E-02-2.4575.86E-0SLITRKS -2.0473.87E-07NSNS-1.1841.54E-06-1.7349.31E-08-1.1521.05E-0SPARCL1 -2.3146.14E-06NSNSNSNS-0.8433.87E-02NSNSTNR -3.0828.83E-06-5.1081.89E-07-2.2278.81E-13NSNS-2.1375.56E-1

[0075] Table 3. Genomic analysis of SCZ-derived hGPCs from 4 different patients revealed the significant and shared down-regulation in these cells of a number of synaptic genes, including neuroligin-3, neuroexophilin-1, LINGO1 and DSCAML1, relative to their normal controls (red, upregulated in SCZ vs CTRL; green, downregulated in SCZ GPCs; color intensity proportionate to differential dysregulation). Other synapse-associated genes, such as the SLITRKs 2-5, were significantly and sharply downregulated in GPCs derived from 3 of the 4 patients (lines 8, 51 and 164). Lines 08, 29, 51 and 164: schizophrenia-derived, different patients; pooled controls, 3 lines, each from a different patient. Individual SCZ line data shown as well as pooled SCZ data, to highlight both commonalities and distinctions between SCZ GPCs derived from different patients. Log2FC: log 2 fold-change in expression. NS: not significant. Whereas the expression of these latter genes was suppressed in hGPCs derived from all 4 SCZ patients, other synapse-associated genes, such as NRXN1, NLGN1, DSCAML1, and the SLITRKs 2-5, were sharply down-regulated in hGPCs derived from 3 of the 4 patients, but not in the fourth (Table 3). Yet other synapse-associated transcripts, like NXPH3 and NTRNG2, were similarly down-regulated 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 validated the significant differential down-regulation of these differentiation and synaptic function-associated genes (Table 4 and Figure 8).

[0076] Together, these data suggest the importance of glial-associated synaptic gene expression in schizophrenia, while emphasizing the heterogeneity of pathways that might be mechanistically complicit in its dysregulation. These data also highlight the point that while the neuronal localization of these synaptic proteins has long been recognized, their synthesis by glia and synaptic contributions thereof have not been specifically discussed, although cell type-specific transcriptional databases have noted significant glial expression of these genes (Zhang et al., "An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex," The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 34:11929-11947 (2014)). Since NRXN1, a synapse-associated transcript closely linked to schizophrenia (Sudhof, T. C., "Neuroligins and Neurexins Link Synaptic Function to Cognitive Disease," Nature 455:903-911 (2008)), was one of the most strongly and consistently down-regulated glial genes across the patients, the down-regulation of its expression by SCZ glia was verified, 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 9). Table 4 Gene Symbol qPCR Ratio (min-max; P Value) Transcription regulators LINGO1***0.105 (0.064 - 0.174; P = 2.20E-03)MYRF0.263 (0.092 - 0.754; P = 1.11E-01)NKX2-2**0.253 (0.115 - 0.556; P = 2.17E-02)OLIG1***0.170 (0.076 - 0.381; P = 4.83E-03)OLIG2***0.119 (0.053 - 0.268; P = 1.09E-03)SOX10***0.049 (0.013 - 0.178; P = 1.09E-03)SOX9*0.692 (0.524 - 0.914; P = 7.76E-02)TCF7L2*0.639 (0.451 - 0.906; P = 7.70E-02)ZNF488**0.130 (0.042 - 0.397; P = 2.12E-02)Myelination-associated CNTN1***0.076 (0.046 - 0.127; P = 9.83E-04)FA2H***0.040 (0.010 - 0.171; P = 1.34E-03)GPR17**0.094 (0.019 - 0.467; P = 2.18E-02)MPZ0.482 (0.211 - 1.103; P = 4.99E-01)OMG***0.154 (0.082 - 0.287; P = 1.09E-03)SIRT2***0.401 (0.307 - 0.524; P = 4.54E-03)UGT8***0.045 (0.012 - 0.160; P = 1.09E-03)Synaptic junction-associated ATP2B2***0.209 (0.113 - 0.385; P = 2.49E-03)BCAN***0.191 (0.113 - 0.323; P = 2.49E-03)CD44**2.397 (1.435 - 4.007; P = 3.19E-02)CRHR1**0.183 (0.058 - 0.583; P = 2.24E-02)DSCAML1***0.302 (0.190 - 0.480; P = 2.49E-03)LGI10.821 (0.228 - 2.957; P = 8.10E-01)NETO1***0.095 (0.042 - 0.214; P = 9.83E-04)NRXN1**0.252 (0.124 - 0.510; P = 2.12E-02)NTNG1**0.348 (0.149 - 0.817; P = 4.69E-02)NTNG20.569 (0.223 - 1.451; P = 3.66E-01)NXPH1***0.085 (0.034 - 0.216; P = 1.30E-03)RPH3A***0.205 (0.130 - 0.324; P = 3.24E-03)SLC6A1***0.188 (0.094 - 0.378; P = 4.44E-03)SLITRK2**0.010 (0.003 - 0.033; P = 3.19E-02)SLITRK3***0.074 (0.025 - 0.220; P = 1.33E-03)SLITRK4*0.101 (0.030 - 0.335; P = 5.73E-02)SPARCL1*0.507 (0.275 - 0.936; P = 9.14E-02)TNR***0.102 (0.045 - 0.228; P = 9.83E-04)Ion channels KCNA3**0.270 (0.166 - 0.439; P = 1.30E-02)KCND2***0.070 (0.025 - 0.196; P = 1.09E-03)KCNH80.534 (0.261 - 1.093; P = 2.56E-01)KCNJ9**0.257 (0.151 - 0.439; P = 2.24E-02)KCNK9**0.159 (0.061 - 0.411; P = 1.28E-02)Actin ACTB1.365 (1.016 - 1.833; P = 1.27E-01)Astrocyte-specific marker GFAP1.479 (0.819 - 2.672; P = 4.25E-01)Enzyme ALDH1L10.596 (0.164 - 2.170; P = 5.67E-01)Growth factor FGF14***0.085 (0.034 - 0.213; P = 1.33E-03)RNA binding protein ELAVL40.585 (0.374 - 0.915; P = 1.61E-01)Wnt signaling WNT7B*0.149 (0.042 - 0.527; P = 5.43E-02)

[0077] Table 4. Expression of selected genes identified by RNA-seq analysis as dysregulated in SCZ-derived GPCs was assessed by TaqMan Low Density Array (TLDA) RT-qPCR, and compared to that of control GPCs. Expression data were normalized to GAPDH endogenous control. Mean expression ratios calculated from 4 pooled SCZ GPC lines (n = 19) against 3 pooled control GPC lines (n = 10) are shown. The difference of expression in SCZ and control GPCs was assessed by paired t-test followed by multiple testing correction by Benjamini-Hochberg (BH) procedure. BH-corrected P values are shown (*** = 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 proportion of undetermined and unreliable reactions, LRFN1 and NEUROD6. The vast majority of genes were confirmed as dysregulated in SCZ-derived GPCs which reliably exhibited the significant differential down-regulation of differentiation, potassium channel and synapse function-associated genes. Analysis of TLDA data was performed in ExpressionSuite Software version 1.1 supplied by Applied Biosciences.Example 5 - SCZ Glial Chimerization Yielded Disease-Specific Behavioral Phenotype

[0078] It was 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, it was postulated that the aberrant infiltration of hGPCs and their derived astroglia into the developing cortex might influence information processing within the cortex once 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 (Oberheim et al., "Uniquely Hominid Features of Adult Human Astrocytes," J. Neurosci. 29:3276-3287 (2009); Han et al., "Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning in Adult Mice," Cell Stem Cell 12:342-353 (2013)). 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, which 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 do allografted or untransplanted controls (Han et al., "Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning in Adult Mice," Cell Stem Cell 12:342-353 (2013)). Thus, engrafted human GPCs and their daughter glia can integrate into, and substantially modify, developing neural networks (Franklin et al., "Do Your Glial Cells Make You Clever?," Cell Stem Cell 12:265-266 (2013)). On that basis, it was postulated that the disruption in normal glial development noted in the SCZ glial chimeras might yield disease-associated changes in learning and behavior. To address this question, the behavioral phenotypes of immunodeficient but otherwise wild-type mice neonatally engrafted with SCZ GPCs were assessed, relative to matched hosts engrafted with control-derived GPCs. For these experiments, normally-myelinated hosts were used rather than shiverer mice, so as 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.

[0079] It was first asked whether schizophrenic derivation of engrafted glia affected prepulse inhibition (PPI), a behavioral hallmark of both clinical schizophrenics and animal models thereof (Ewing et al., "Evidence for Impaired Sound Intensity Processing During Prepulse Inhibition of the Startle Response in a Rodent Developmental Disruption Model of Schizophrenia," Journal of Psychiatric Research (2013)). PPI reflects the coordination of sensorimotor gating in the CNS, and its diminution may predict aspects of schizophrenic phenotype (Ivleva et al., "Smooth Pursuit Eye Movement, Prepulse Inhibition, and Auditory Paired Stimuli Processing Endophenotypes Across the schizophrenia-Bipolar Disorder Dimension," Schizophrenia Bulletin (2013); Kohl et al., "Prepulse Inhibition in Psychiatric Disorders--Apart from Schizophrenia," Journal of Psychiatric Research 47:445-452 (2013)). It was found that when assessed at 6 months of age - the latest time-point at which the C57Bl / 6 background strain of the rag1 - / -< mice can be reliably assessed, since these mice suffer premature auditory loss which might otherwise diminish auditory PPI - that mice engrafted with SCZ hGPCs exhibited significantly diminished auditory prepulse inhibition (Figure 10A), and did so at all volumes of pre-pulse. Given the strong effect of SCZ glial chimerization on PPI, it was next asked if SCZ glial chimerization might be associated with changes in behavior on cognitive and socialization tests. To that end, SCZ and control chimeras were compared on a battery of behavioral tests that included: 1) the elevated plus maze, a measure of anxiety (Walf et al., "The Use of the Elevated Plus Maze as an Assay of Anxiety-Related Behavior in Rodents," Nat Protoc 2:322-328 (2007)); 2) the 3-chamber social challenge (Yang et al., "Automated Three-Chambered Social Approach Task for Mice," Curr Protoc Neurosci, Chapter 8, Unit 8, 26 (2011)); 3) novel object recognition, a focused measure of executive memory (Bevins et al., "Object Recognition in Rats and Mice: A One-Trial Non-Matching-to-Sample Learning Task to Study 'Recognition Memory'," Nat Protoc 1:1306-1311 (2006)), and 4) the preference for sucrose water, a test for anhedonia (Barnes et al., "Anhedonia, Avolition, and Anticipatory Deficits: Assessments in Animals with Relevance to the Negative Symptoms of Schizophrenia," Eur Neuropsychopharmacol 24:744-758 (2014); Willner et al., "Reduction of Sucrose Preference by Chronic Unpredictable Mild Stress, and its Restoration by a Tricyclic Antidepressant," Psychopharmacology (Berl) 93:358-364 (1987)). In each, mice chimerized with one of 3 SCZ or 3 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. Normal control-engrafted mice are significantly more likely to explore the open arms (horizontal segments), whereas SCZ mice spent most of their time in the closed maze arms (vertical segments), consistent with greater anxiety (p=0.036, 2-tailed t test). The SCZ hGPC mice exhibited greater avoidance of the open arms in the elevated plus maze than did their normal hGPC-engrafted controls (n=36 mice / group, each including 12 mice engrafted with hGPCs from each of 3 patients; p=0.036, 2-tailed t test), suggesting that the SCZ hGPC mice were prone to higher anxiety when challenged (Figure 10B). In addition, the SCZ hGPC mice showed less preference for sucrose water), consistent with relative anhedonia (Figure 10C), less interest in stranger mice in the 3-chamber social test (Figure 10D), and relatively poor novel object recognition (Figure 10E), reflecting relative impairment in executive memory.

[0080] As an additional metric of SCZ-associated behavior, sleep and diurnal activity patterns of human SCZ and CTRL glial chimeras were then assessed, directly comparing mice engrafted with either SCZ (line 52) or matched control (line 22) hGPCs. It was 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-hour video-recording (Noldus Ethovision), the SCZ hGPC chimeric mice moved significantly more than their normal hGPC-engrafted controls (2-way ANOVA, F=48.35; p<0.0001) (Figure 10F). Interestingly, while the SCZ-associated increment in activity largely occurred during night-time periods of wakefulness, the SCZ mice also manifested disrupted sleep patterns, as measured by the duration of bouts of inactivity, a surrogate for EEG-validated sleep (Pack et al., "Novel Method for High-Throughput Phenotyping of Sleep in Mice," Physiol. Genomics 28:232-238 (2007) ; McShane et al., "Characterization of the Bout Durations of Sleep and Wakefulness," J. Neurosci. Methods 193:321-333 (2010),) (Figure 10G). Within the half-hour following the phase transition from dark to light (when mice normally sleep), the CTRL mice had more continuous, uninterrupted patterns of sleep, with an average sleep bout of 511.5 ± 36.4 seconds (8.53 minutes), whereas SCZ mice were asleep for 306.2 ± 43.7 seconds, or 5.1 minutes per bout (p<0.01 by 2-way ANOVA, with Boneferroni post hoc t tests). The shorter average periods of inactivity manifested by SCZ hGPC mice during the normal daytime transition to sleep suggests that SCZ hGPC chimerization disrupted normal daytime sleep patterns, while increasing night-time 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 schizophrenia.Discussion of Examples

[0081] These data suggest a significant contribution of cell-autonomous glial pathology to the genesis and development of juvenile-onset schizophrenia. In these human glial chimeric mice, schizophrenia-derived iPSC hGPCs exhibited aberrant migration with deficient engraftment in the central white matter, relative to age and gender-matched control iPSC hGPCs. Although a fraction of those SCZ hGPCs that did remain within the white matter differentiated as normal myelinogenic oligodendroglia, the premature cortical influx and hence lower density of donor-derived cells in the white matter of SCZ hGPC-engrafted mice resulted in the latter's overt hypomyelination, relative to mice engrafted with control GPCs. Thus, SCZ hGPCs appeared to traverse rather than home in to the nascent white matter, resulting in sparse hGPC colonization and hence deficient forebrain myelination. The aberrant dispersal pattern of SCZ hGPCs suggests that SCZ GPCs may not recognize developmental stop signals that permit progenitors to dwell and expand within the presumptive white matter before colonizing the cortical mantle, and may instead be biased towards rapid entry into the cortical gray matter. These observations in human SCZ glial chimeric mice are especially intriguing given the well-described hypomyelination of schizophrenic patients (Voineskos et al., "Oligodendrocyte Genes, White Matter Tract Integrity, and Cognition in Schizophrenia," Cereb Cortex 23:2044-2057 (2013); Najjar et al., "Neuroinflammation and White Matter Pathology in Schizophrenia: Systematic Review," Schizophrenia Research 161:102-112 (2015); Davis et al., "White Matter Changes in Schizophrenia: Evidence for Myelin-Related Dysfunction," Archives of General Psychiatry 60:443-456 (2003); Sigmundsson et al., "Structural Abnormalities in Frontal, Temporal, and Limbic Regions and Interconnecting White Matter Tracts in Schizophrenic Patients with Prominent Negative Symptoms," Am J Psychiatry 158:234-243 (2001)), particularly so in early onset disease (Gogtay et al., "Three-Dimensional Brain Growth Abnormalities in Childhood-Onset Schizophrenia Visualized by Using Tensor-Based Morphometry," Proceedings of the National Academy of Sciences of the United States of America 105:15979-15984 (2008); Samartzis et al., "White Matter Alterations in Early Stages of Schizophrenia: A Systematic Review of Diffusion Tensor Imaging Studies," J Neuroimaging 24:101-110 (2014); Gogtay et al., "Childhood-Onset Schizophrenia: Insights From Neuroimaging Studies," Journal of the American Academy of Child and Adolescent Psychiatry 47:1120-1124 (2008)).

[0082] These anatomic observations were especially intriguing in light of the differential gene expression pattern of the SCZ hGPCs, which revealed that the cells were deficient not only in early glial differentiation-associated transcripts, but also in genes that encode for synaptic proteins typically associated with transducing activity-dependent signals (Sudhof, T. C., "Neuroligins and Neurexins Link Synaptic Function to Cognitive Disease," Nature 455:903-911 (2008)). Together, these anatomic and transcriptional data suggest that SCZ hiPSC-derived GPCs might be subject to impaired phenotypic differentiation, that might result in their neglect of the local neuronal signals that typically regulate the expansion and maturation of GPCs (Barres et al., "Proliferation of Oligodendrocyte Precursor Cells Depends on Electrical Activity in Axons," Nature 361:258-260 (1993)); this might account for their rapid transit through the white matter into the overlying cortex, and hence the diminished callosal GPC density and hypomyelination of SCZ chimeric shiverer mice (Figure 3). Thus, the myelination defect in SCZ hGPC chimeras appeared due to both deficient oligodendrocytic differentiation and the relative dearth of SCZ hGPCs remaining within the white matter. Moreover, astrocytic differentiation from SCZ hGPCs was also impaired, and may have contributed further to hypomyelination in the SCZ glial chimeras, given the metabolic dependence of mature oligodendrocytes upon local astrocytes (Amaral et al., "Metabolic Aspects of Neuron-Oligodendrocyte-Astrocyte Interactions," Front Endocrinol (Lausanne) 4:54 (2013); John, G. R., "Investigation of Astrocyte - Oligodendrocyte Interactions in Human Cultures," Methods Mol Biol 814:401-414 (2012)).

[0083] Importantly, the defective astrocytic maturation of SCZ hGPCs might also have profound effects on developmental synaptogenesis and circuit formation, as well as on myelinogenesis. Neural connectivity and synaptic development are both intimately dependent upon astrocytic guidance (Clarke et al., "Glia Keep Synapse Distribution Under Wraps," Cell 154:267-268 (2013); Ullian et al., "Control of Synapse Number by Glia," Science 291:657-661 (2001)), and hence upon the appropriate timing of astrocytic appearance and maturation. As a result, any disruption in astrocytic maturation by SCZ hGPCs, as observed in each of the SCZ lines studied, might be expected to significantly confound the construction and functional architecture of those neural networks in which SCZ hGPCs are resident. Moreover, glial progenitors themselves may have significant interactions with local neurons (Sakry et al., "Oligodendrocyte Precursor Cells Modulate the Neuronal Network by Activity-Dependent Ectodomain Cleavage of Glial NG2," PLoS Biol 12:e1001993 (2014)), such that their dysfunction might disrupt local neuronal response thresholds and circuit formation.

[0084] Besides the anatomic observation of deficient astrocytic maturation in SCZ hGPC chimeras, the genomic analysis of SCZ-derived hGPCs revealed the significant down-regulation in hGPCs derived from all 4 SCZ patients of a number of synaptic genes, including neuroligin-3, neuroexophilin-1, and LINGO1 relative to their normal controls (Tables 3 and Table 4; Figure 8). Other synapse-associated genes, such as neurexin-1 and DSCAML1 were significantly and sharply down-regulated in GPCs derived from 3 patients (lines 8, 29, and 51) but not in the fourth (line 164). Similarly, SLITRKs 2-5 were significantly and sharply down-regulated in GPCs derived from 3 patients (lines 8, 51, and 164), but not in a fourth (line 29), which was instead associated with sharp down-regulation of LINGO1, DSCAML1, and several neurexins and neuroexophilins; these data suggesting the heterogeneity of transcriptional dysfunction that may lead to a final common pathway of glial-involved synaptic dysfunction in SCZ (Tables 2 and 3). These transcripts are critical contributors to synaptic stabilization and function (Sudhof, T. C., "Neuroligins and Neurexins Link Synaptic Function to Cognitive Disease," Nature 455:903-911 (2008)), but while typically considered neuronal, may be produced significantly by glial cells as well (Zhang et al., "An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex," J. Neurosci. 34:11929-11947 (2014)). The relative down-regulation 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 (De Biase et al., "Excitability and Synaptic Communication Within the Oligodendrocyte Lineage," J Neurosci 30:3600-3611 (2010); Lin et al., "Synaptic Signaling Between GABAergic Interneurons and Oligodendrocyte Precursor Cells in the Hippocampus," Nat. Neurosci. 7:24-32 (2004)). 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.

[0085] Schizophrenia is genetically heterogeneous, so that 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 3 different SCZ patients were thus compared anatomically to those established from GPCs derived from 3 control patients. None of the controls manifested the white matter-avoidant dispersal pattern of the SCZ hGPC chimeras. Similarly, this pattern of SCZ hGPC avoidance of the white matter had never been noted in any of several hundred human glial chimeras engrafted in other studies with either fetal tissue-derived (Windrem et al., "Neonatal Chimerization with Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008); Windrem et al., "A Competitive Advantage by Neonatally Engrafted Human Glial Progenitors Yields Mice Whose Brains are Chimeric for Human Glia," J. Neurosci. 34:16153-16161 (2014)) or normal iPSC-derived (Wang et al., "Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination," Cell Stem Cell 12:252-264 (2013)) hGPCs.

[0086] Besides their clear anatomic phenotype, the SCZ hGPC-chimeric mice manifested robust behavioral phenotypes. They exhibited significantly attenuated prepulse inhibition relative to control-engrafted mice, relative anhedonia, excessive anxiety, deficient socialization with avoidance of conspecifics, and disrupted patterns of diurnal activity and sleep. These data establish that SCZ glial engraftment may yield an abnormal behavioral phenotype in recipient mice, along behavioral axes that typify selected aspects of schizophrenic behavioral pathology in humans. In that regard, while an extensive literature has implicated GPCs (De Biase et al., "Excitability and Synaptic Communication Within the Oligodendrocyte Lineage," J Neurosci 30:3600-3611 (2010); Bergles et al., "Neuron-Glia Synapses in the Brain," Brain Res Rev 63:130-137 (2010)) as well as astroglia (Kang et al., "Astrocyte-Mediated Potentiation of Inhibitory Synaptic Transmission," Nature Neuroscience 1:683-692 (1998); Araque et al., "Glutamate-Dependent Astrocyte Modulation of Synaptic Transmission Between Cultured Hippocampal Neurons," European J. Neurosci. 10 (1998)) in the modulation of synaptic plasticity and learning (Han et al., "Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning in Adult Mice," Cell Stem Cell 12:342-353 (2013)), these data do not implicate one phenotype over the other in the modulation of behavior by SCZ glial chimerization; the chimeric mice are colonized by both donor-derived human GPCs and their derived astrocytes. That said, the observations of significant defects in SCZ glial maturation shared by hGPCs derived from multiple independent patients, associated in each with hypomyelination and disrupted astrocytic differentiation, as well as with abnormal behavioral phenotypes in the resultant SCZ GPC chimeras, together suggest a strong causal contribution of glial pathology to schizophrenia. In addition, these data highlight the potential of disease-specific humanized chimeras in defining the respective contributions of glial and neuronal dysfunction in the genesis and course of neurological disease.

Claims

1. A preparation of glial progenitor cells for use in treating schizophrenia in a subject, wherein said preparation of glial progenitor cells is administered to a subject having schizophrenia at a dosage effective to treat schizophrenia in the subject.

2. The preparation for use according to Claim 1, wherein the glial progenitor cells of the preparation are A2B5+, CD140a+, and / or CD44+.

3. The preparation for use according to Claim 1, wherein the administration is intracerebral, intraventricular, intrathecal or intracisternal administration.

4. The preparation for use according to Claim 1, wherein the subject is human.

5. The preparation for use according to Claim 1, wherein the glial progenitor cells are able to produce astrocytes.

6. The preparation for use according to Claim 1, wherein the glial progenitor cells are derived from fetal tissue, embryonic stem cells, or induced pluripotent stem cells.

7. The preparation for use according to Claim 1, wherein the preparation of glial progenitor cells is a preparation of human glial progenitor cells.

8. The preparation for use according to Claim 1, wherein the glial progenitor cells are able to produce oligodendrocytes.