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Glial pathology is a causal contributor to the striatal neuronal dysfunction of Huntington's disease (HD). We investigate mutant HTT-associated changes in gene expression by mouse and human striatal astrocytes, as well as in mouse microglia, to identify commonalities in glial pathobiology across species and models. Mouse striatal astrocytes are fluorescence-activated cell sorted (FACS) from R6/2 and zQ175 mice, which respectively express exon1-only or full-length mHTT, and human astrocytes are generated either from human embryonic stem cells (hESCs) expressing full-length mHTT or from fetal striatal astrocytes transduced with exon1-only mHTT. Comparison of differential gene expression across these conditions, all with respect to normal HTT controls, reveals cell-type-specific changes in transcription common to both species, yet with differences that distinguish glia expressing truncated mHTT versus full-length mHTT. These data indicate that the differential gene expression of glia expressing truncated mHTT may differ from that of cells expressing full-length mHTT, while identifying a conserved set of dysregulated pathways in HD glia.

Summary: Neonatally transplanted human glial progenitor cells (hGPCs) can myelinate the brains of myelin-deficient shiverer mice, rescuing their phenotype and survival. Yet, it has been unclear whether implanted hGPCs are similarly able to remyelinate the diffusely demyelinated adult CNS. We, therefore, ask if hGPCs could remyelinate both congenitally hypomyelinated adult shiverers and normal adult mice after cuprizone demyelination. In adult shiverers, hGPCs broadly disperse and differentiate as myelinating oligodendrocytes after subcortical injection, improving both host callosal conduction and ambulation. Implanted hGPCs similarly remyelinate denuded axons after cuprizone demyelination, whether delivered before or after demyelination. RNA sequencing (RNA-seq) of hGPCs back from cuprizone-demyelinated brains reveals their transcriptional activation of oligodendrocyte differentiation programs, while distinguishing them from hGPCs not previously exposed to demyelination. These data indicate the ability of transplanted hGPCs to disperse throughout the adult CNS, to broadly myelinate regions of dysmyelination, and also to be recruited as myelinogenic oligodendrocytes later in life, upon demyelination-associated demand.

As new methods for producing and isolating human glial progenitor cells (hGPCs) have been developed, the disorders of myelin have become especially compelling targets for cell-based therapy. Yet as animal modeling of glial progenitor cell-based therapies has progressed, it has become clear that transplanted hGPCs not only engraft and expand within murine hosts, but dynamically outcompete the resident progenitors so as to ultimately dominate the host brain. The engrafted human progenitor cells proceed to generate parenchymal astrocytes, and when faced with a hypomyelinated environment, oligodendrocytes as well. As a result, the recipient brains may become inexorably humanized with regards to their resident glial populations, yielding human glial chimeric mouse brains. These brains provide us a fundamentally new tool by which to assess the species-specific attributes of glia in modulating human cognition and information processing. In addition, the cellular humanization of these brains permits their use in studying glial infectious and inflammatory disorders unique to humans, and the effects of those disorders on the glial contributions to cognition. Perhaps most intriguingly, by pairing our ability to construct human glial chimeras with the production of patient-specific hGPCs derived from pluripotential stem cells, we may now establish mice in which a substantial proportion of resident glia are both human and disease-derived. These mice in particular may provide us new opportunities for studying the human-specific contributions of glia to human psychopathology, as well as to higher cognition. As such, the assessment of human glial chimeric mice may provide us new insight into the species-specific contributions of glia to human cognitive evolution, as well as to the pathogenesis of human neurological and neuropsychiatric disease.

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

The causal contribution of glial pathology to Huntington disease (HD) has not been heavily explored. To define the contribution of glia to HD, we established human HD glial chimeras by neonatally engrafting immunodeficient mice with mutant huntingtin (mHTT)-expressing human glial progenitor cells (hGPCs), derived from either human embryonic stem cells or mHTT-transduced fetal hGPCs. Here we show that mHTT glia can impart disease phenotype to normal mice, since mice engrafted intrastriatally with mHTT hGPCs exhibit worse motor performance than controls, and striatal neurons in mHTT glial chimeras are hyperexcitable. Conversely, normal glia can ameliorate disease phenotype in transgenic HD mice, as striatal transplantation of normal glia rescues aspects of electrophysiological and behavioural phenotype, restores interstitial potassium homeostasis, slows disease progression and extends survival in R6/2 HD mice. These observations suggest a causal role for glia in HD, and further suggest a cell-based strategy for disease amelioration in this disorder.

Experimental animals with myelin disorders can be treated by transplanting oligodendrocyte progenitor cells (OPCs) into the affected brain or spinal cord. OPCs have been isolated by their expression of gangliosides recognized by mAb A2B5, but this marker also identifies lineage-restricted astrocytes and immature neurons. To establish a more efficient means of isolating myelinogenic OPCs, we sorted fetal human forebrain cells for CD140a, an epitope of platelet derived growth factor receptor (PDGFR)α, which is differentially expressed by OPCs. CD140a(+) cells were isolated as mitotic bipotential progenitors that initially expressed neither mature neuronal nor astrocytic phenotypic markers, yet could be instructed to either oligodendrocyte or astrocyte fate in vitro. Transplanted CD140a(+) cells were highly migratory and robustly myelinated the hypomyelinated shiverer mouse brain more rapidly and efficiently than did A2B5(+)cells. Microarray analysis of CD140a(+) cells revealed overexpression of the oligodendroglial marker CD9, suggesting that CD9(+)/CD140a(+) cells may constitute an even more highly enriched population of myelinogenic progenitor cells.

Both late-gestation and adult human forebrain contain large numbers of oligodendrocyte progenitor cells (OPCs). These cells may be identified by their A2B5(+)PSA-NCAM(-) phenotype (positive for the early oligodendrocyte marker A2B5 and negative for the polysialylated neural cell adhesion molecule). We used dual-color fluorescence-activated cell sorting (FACS) to extract OPCs from 21- to 23-week-old fetal human forebrain, and A2B5 selection to extract these cells from adult white matter. When xenografted to the forebrains of newborn shiverer mice, fetal OPCs dispersed throughout the white matter and developed into oligodendrocytes and astrocytes. By 12 weeks, the host brains showed extensive myelin production, compaction and axonal myelination. Isolates of OPCs derived from adult human white matter also myelinated shiverer mouse brain, but much more rapidly than their fetal counterparts, achieving widespread and dense myelin basic protein (MBP) expression by 4 weeks after grafting. Adult OPCs generated oligodendrocytes more efficiently than fetal OPCs, and ensheathed more host axons per donor cell than fetal cells. Both fetal and adult OPC phenotypes mediated the extensive and robust myelination of congenitally dysmyelinated host brain, although their differences suggested their use for different disease targets.

Neonatally transplanted human glial progenitor cells (hGPCs) densely engraft and myelinate the hypomyelinatedshiverermouse. We found that, in hGPC-xenografted mice, the human donor cells continue to expand throughout the forebrain, systematically replacing the host murine glia. The differentiation of the donor cells is influenced by the host environment, such that more donor cells differentiated as oligodendrocytes in the hypomyelinatedshivererbrain than in myelin wild-types, in which hGPCs were more likely to remain as progenitors. Yet in each recipient, both the number and relative proportion of mouse GPCs fell as a function of time, concomitant with the mitotic expansion and spread of donor hGPCs. By a year after neonatal xenograft, the forebrain GPC populations of implanted mice were largely, and often entirely, of human origin. Thus, neonatally implanted hGPCs outcompeted and ultimately replaced the host population of mouse GPCs, ultimately generating mice with a humanized glial progenitor population. These human glial chimeric mice should permit us to define the specific contributions of glia to a broad variety of neurological disorders, using human cellsin vivo.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease triggered by infection with the human gliotropic JC virus (JCV). Due to the human-selective nature of the virus, there are no animal models available to investigate JCV pathogenesis. To address this issue, we developed mice with humanized white matter by engrafting human glial progenitor cells (GPCs) into neonatal immunodeficient and myelin-deficient mice. Intracerebral delivery of JCV resulted in infection and subsequent demyelination of these chimeric mice. Human GPCs and astrocytes were infected more readily than oligodendrocytes, and viral replication was noted primarily in human astrocytes and GPCs rather than oligodendrocytes, which instead expressed early viral T antigens and exhibited apoptotic death. Engraftment of human GPCs in normally myelinated and immunodeficient mice resulted in humanized white matter that was chimeric for human astrocytes and GPCs. JCV effectively propagated in these mice, which indicates that astroglial infection is sufficient for JCV spread. Sequencing revealed progressive mutation of the JCV capsid protein VP1 after infection, suggesting that PML may evolve with active infection. These results indicate that the principal CNS targets for JCV infection are astrocytes and GPCs and that infection is associated with progressive mutation, while demyelination is a secondary occurrence, following T antigen-triggered oligodendroglial apoptosis. More broadly, this study provides a model by which to further assess the biology and treatment of human-specific gliotropic viruses.

The diseases of myelin are among the most prevalent and disabling conditions in neurology. These diseases include both the vascular and inflammatory demyelinating disorders of adulthood, as well as the childhood leukodystrophies and cerebral palsy. These fundamentally glial disorders may be amenable to treatment by glial progenitor cells (GPCs), which give rise to astroglia and myelin-producing oligodendrocytes. Given the development of new methods for generating and isolating human GPCs, the myelin disorders may now be compelling targets for cell-based therapy. In addition, the efficient engraftment and expansion of human GPCs in murine hosts has led to the development of human glial chimeric mouse brains, which provides new opportunities for studying the species-specific roles of human glia in cognition, as well as in disease pathogenesis.

Glial progenitor cells (GPCs) comprise the most abundant population of progenitor cells in the adult human brain. They are responsible for central nervous system (CNS) remyelination, and likely contribute to the astrogliotic response to brain injury and degeneration as well. Adult human GPCs are biased to differentiate as oligodendrocytes and elaborate new myelin, and yet they retain multilineage plasticity, and can give rise to neurons as well as astrocytes and oligodendrocytes once removed from the adult parenchymal environment. GPCs retain strong mechanisms for cell-autonomous self-renewal, and yet both their phenotype and fate may be dictated by their microenvironment. Using the transcriptional profiles of acutely isolated GPCs, we have begun to understand the operative ligand–receptor interactions involved in these processes, and have identified several key signaling pathways by which adult human GPCs may be reliably instructed to either oligodendrocytic or astrocytic fate. In addition, we have noted significant differences between the expressed genes and dominant signaling pathways of fetal and adult human GPCs, as well as between rodent and human GPCs. The latter data in particular call into question therapeutic strategies predicated solely upon data obtained using rodents, while perhaps highlighting the extent to which evolution has been attended by the phylogenetic modification of glial phenotype and function.

The diversity of neocortical cytoarchitecture could arise from genetic prespecification of cell types and numbers in the ventricular zone, by interaction of cells with their immediate environment, efferent targets, or afferent inputs. Here, we examine the role of the thalamus as efferent target or afferent source in the early control of cell number and type in the developing neocortex. Electrolytic lesions of the thalamus were made in hamsters at birth prior to the thalamic innervation of layer IV. By postnatal day 7, when migration to the cortex is complete, there were no differences in cell number between the cortical plate contralateral and ipsilateral to the thalamic lesion, showing that the absence of thalamic input does not influence the last phases of neocortical cell generation or migration. However, the incidence of pyknotic cells was elevated in the upper half of the cortical plate at this time. By adulthood, the number of cells per unit column of cortex was reduced, due to the apparent absence of small, nonpyramidal cells of layer IV, as determined from Nissl-stained material. Therefore, some of the cytoarchitectonic variability of the neocortex could arise epigenetically by the interaction of neocortical cells with their afferent connections.

Summary Congenitally hypomyelinated shiverer mice fail to generate compact myelin and die by 18–21 weeks of age. Using multifocal anterior and posterior fossa delivery of sorted fetal human glial progenitor cells into neonatal shiverer × rag2 −/− mice, we achieved whole neuraxis myelination of the engrafted hosts, which in a significant fraction of cases rescued this otherwise lethal phenotype. The transplanted mice exhibited greatly prolonged survival with progressive resolution of their neurological deficits. Substantial myelination in multiple regions was accompanied by the acquisition of normal nodes of Ranvier and transcallosal conduction velocities, ultrastructurally normal and complete myelination of most axons, and a restoration of a substantially normal neurological phenotype. Notably, the resultant mice were cerebral chimeras, with murine gray matter but a predominantly human white matter glial composition. These data demonstrate that the neonatal transplantation of human glial progenitor cells can effectively treat disorders of congenital and perinatal hypomyelination.

The homeobox gene Hb9 is expressed selectively by motor neurons (MNs) in the developing CNS. Previous studies have identified a 9-kb 5′ fragment of the mouse Hb9 gene that is sufficient to direct gene expression to spinal MNs in vivo. Here, we sought to identify more discrete MN-specifying elements, using homology searches between genomic sequences of evolutionarily distant species. Based on homology screening of the mouse and human Hb9 promoters, we identified a 3.6-kb Hb9 enhancer that proved sufficient to drive MN-specific lacZ expression. We then compared mouse, human, and pufferfish (Fugu rubripes) genomic sequences, and identified a conserved 438-bp sequence, consisting of noncontiguous 313-bp and 125-bp fragments, residing within the 3.6-kb Hb9 enhancer. The zebrafish (Danio rerio) Hb9 genomic region was then found to have two identical copies of the 125-bp sequence, but no counterpart for the 313-bp sequence. Transgenic analysis showed that the 125-bp alone was both necessary and sufficient to direct spinal MN-specific lacZ expression, whereas the 313-bp sequence had no such enhancer activity. Moreover, the 125-bp Hb9 enhancer was found to harbor two Hox/Pbx consensus-binding sequences, mutations of which completely disrupted thoracolumbar Hb9 expression. These data suggest that Hox/Pbx plays a critical role in the segmental specification of spinal MNs. Together, these results indicate that the molecular pathways regulating Hb9 expression are evolutionarily conserved, and that MN-specific gene expression may be directed and achieved using a small 125-bp 5′ enhancer.

A distinct population of white matter progenitor cells (WMPCs), competent but not committed to generate oligodendrocytes, remains ubiquitous in the adult human subcortical white matter. These cells are present in both sexes and into senescence and may constitute as much as 4% of the cells of adult human capsular white matter. Transduction of adult human white matter dissociates with plasmids bearing early oligodendrocytic promoters driving fluorescent reporters permits the separation of these cells at high yield and purity, as does separation based on their expression of A2B5 immunoreactivity. Isolates of these cells survive xenograft to lysolecithin-demyelinated brain and migrate rapidly to infiltrate these lesions, without extending into normal white matter. Within several weeks, implanted progenitors mature as oligodendrocytes, and develop myelin-associated antigens. Lentiviral tagging with green fluorescent protein confirmed that A2B5-sorted progenitors develop myelin basic protein expression within regions of demyelination and that they fail to migrate when implanted into normal brain. Adult human white matter progenitor cells can thus disperse widely through regions of experimental demyelination and are able to differentiate as myelinating oligodendrocytes. This being the case, they may constitute appropriate vectors for cell-based remyelination strategies.

The effects of potential excess innervation on cell survival in the superior colliculus and related structures during the period of normally occurring cell death was examined. A unilateral, partial lesion of the superficial layers of the superior colliculus on the day of birth, which results in a compression of the retinotectal map into the remaining area, was the manipulation used to produce the potential excess innervation. Cell density was reduced in the tectal fragment early in development, consistent with hyperinnervation, but had returned to normal by the end of the period of normally occurring cell death. The overall incidence of cell degeneration in the remaining partial colliculus was not different from the undamaged contralateral colliculus or from normal, though there was evidence of a transitory depression and later elevation of cell loss. Cell loss in the retina contralateral to the lesion was increased in the late part of the period of normal cell loss and there were fewer cells in the retinal ganglion cell layer at maturity. The amount of the cell loss in the retina was small compared to the amount of target removal. These results suggest that the survival of neurons with branching axons does not sensitively reflect target availability.

The effect of unilateral deletion of the visual cortex on early cell death and eventual cell number in various structures of the visual system was examined. At minimum, this manipulation potentially provides excess retinal afference to the superior colliculi, partially denervates the superior colliculi, reduces normal retinal terminal area and opens up potential target space for the retina and superior colliculus in those areas where they share terminal space with the visual cortex. All layers of the superior colliculus, bilaterally, showed an initial decrease in the rate of cell death relative to normal followed by an increase in cell death rates. No change in the number or distribution of cells in the retinal ganglion cell layer resulted despite a substantial loss of retinal terminal area, and a substantial alteration of the pattern of retinal central termination. These results are interpreted as evidence for two stages in normally occurring cell death, a first in which axons compete to colonize any available terminal space, and a second in which axon-to-target specificity must be matched. These results also provide evidence that the amount of target required for neuron survival is clearly variable.

To determine if cell death participates in the regulation of cell number between interconnecting populations of the neocortex, we sectioned the corpus callosum of neonatal hamsters, thus depriving callosally projecting cells of their normal targets and callosally-recipient cells of their normal afference. The numbers of neurons per unit column in two areas of the cortex which have heavy callosal projections (the 17-18a border and area 6) and one area that is relatively acallosal (area 3) were compared in animals with early corpus callosum sections and controls. No differences were found, either for a 'unit cortical column,' or for the callosally-projecting layers (II-III and V). Mean soma sizes in layers II-III and V of all three areas were likewise unchanged. In area 6 and part of area 3, however, the distribution of soma sizes in callosally projecting and recipient laminae was significantly altered. The change in size distribution without change in mean soma area suggests that the cortex responds to the elimination of the callosal pathway in more than one way. Since no role for cell death in removal of diffuse connectivity or in target regulation of neuron number has yet been found, a new hypothesis for the function of cell death in local cytoarchitectural differentiation of the cortex is proposed.

In hamster, following removal of one eye on the day of birth the amount of normally occurring degeneration in the retinal ganglion cell layer of the remaining eye is reduced, particularly in the temporal retina. To examine the changes in the number and distribution of cells indicated by the alterations in early cell degeneration, adult retinas from hamsters who had one eye removed at birth were compared to those of normal adults. Normal adult retinal ganglion cell layers were found to contain an average of 157,223 cells (a population which includes retinal ganglion cells, "displaced" amacrine cells, and glia). At adulthood, the remaining retinas of early enucleates had an average of 169,863 cells in the ganglion cell layer, an increase of 8%. If only cells having Nissl substance and a soma diameter in excess of 10 micron (a group likely to consist entirely of retinal ganglion cells) are considered, the increase observed was 19%. Cells having Nissl substance and soma diameters between 5 and 10 micron, a group which includes both retinal ganglion cells and displaced amacrines, show a 13% change. Cells less than 10 micron with no Nissl substance visible, which include displaced amacrines and glial cells showed no net change (75,883 versus 75,409). The increase in cell number was found across the entire retina, but was largest in the temporal retina. These results show that alteration in early neuronal survival is a component of early plastic changes in the central nervous system, and that early cell degeneration rates are good predictors of later cell number and distribution.