JC virus spread is potentiated by glial replication and demyelination-linked glial proliferation.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating infection of the immunosuppressed brain, mediated by the gliotropic polyomavirus JCV. JCV replicates in human glial progenitor cells and astrocytes, which undergo viral T antigen-triggered mitosis, enabling viral replication. We asked if JCV spread might therefore be accelerated by glial proliferation. Using both in vitro analysis and a human glial chimeric mouse model of JCV infection, we found that dividing human astrocytes supported JCV propagation to a substantially greater degree than did mitotically quiescent cells. Accordingly, bulk and single cell RNA-sequence analysis revealed that JCV-infected glia differentially manifested cell cycle-linked disruption of both DNA damage response and transcriptional regulatory pathways. In vivo, JCV infection of humanized glial chimeras was greatly accentuated by cuprizone-induced demyelination and its associated mobilization of GPCs. Importantly, in vivo infection triggered the death of uninfected as well as infected glia, reflecting significant bystander death. Together, these data suggest that JCV propagation in PML may be accelerated by glial cell division. As such, the accentuated glial proliferation attending disease-associated demyelination may provide an especially favorable environment for JCV propagation, thus potentiating oligodendrocytic bystander death and further accelerating demyelination in susceptible hosts.

Modeling the Mutational and Phenotypic Landscapes of Pelizaeus-Merzbacher Disease with Human iPSC-Derived Oligodendrocytes.

Pelizaeus-Merzbacher disease (PMD) is a pediatric disease of myelin in the central nervous system and manifests with a wide spectrum of clinical severities. Although PMD is a rare monogenic disease, hundreds of mutations in the X-linked myelin gene proteolipid protein 1 (PLP1) have been identified in humans. Attempts to identify a common pathogenic process underlying PMD have been complicated by an incomplete understanding of PLP1 dysfunction and limited access to primary human oligodendrocytes. To address this, we generated panels of human induced pluripotent stem cells (hiPSCs) and hiPSC-derived oligodendrocytes from 12 individuals with mutations spanning the genetic and clinical diversity of PMD-including point mutations and duplication, triplication, and deletion of PLP1-and developed an in vitro platform for molecular and cellular characterization of all 12 mutations simultaneously. We identified individual and shared defects in PLP1 mRNA expression and splicing, oligodendrocyte progenitor development, and oligodendrocyte morphology and capacity for myelination. These observations enabled classification of PMD subgroups by cell-intrinsic phenotypes and identified a subset of mutations for targeted testing of small-molecule modulators of the endoplasmic reticulum stress response, which improved both morphologic and myelination defects. Collectively, these data provide insights into the pathogeneses of a variety of PLP1 mutations and suggest that disparate etiologies of PMD could require specific treatment approaches for subsets of individuals. More broadly, this study demonstrates the versatility of a hiPSC-based panel spanning the mutational heterogeneity within a single disease and establishes a widely applicable platform for genotype-phenotype correlation and drug screening in any human myelin disorder.

Concise Review: Stem Cell-Based Treatment of Pelizaeus-Merzbacher Disease.

Pelizaeus-Merzbacher disease (PMD) is an X-linked disorder caused by mutation in the proteolipid protein-1 (PLP1) gene, which encodes the proteolipid protein of myelinating oligodendroglia. PMD exhibits phenotypic variability that reflects its considerable genotypic heterogeneity, but all forms of the disease result in central hypomyelination, associated in most cases with early neurological dysfunction, progressive deterioration, and ultimately death. PMD may present as a connatal, classic and transitional forms, or as the less severe spastic paraplegia type 2 and PLP-null phenotypes. These disorders are most often associated with duplications of the PLP1 gene, but can also be caused by coding and noncoding point mutations as well as full or partial deletion of the gene. A number of genetically-distinct but phenotypically-similar disorders of hypomyelination exist which, like PMD, lack any effective therapy. Yet as relatively pure CNS hypomyelinating disorders, with limited involvement of the PNS and relatively little attendant neuronal pathology, PMD and similar hypomyelinating disorders are attractive therapeutic targets for neural stem cell and glial progenitor cell transplantation, efforts at which are now underway in a number of research centers. Stem Cells 2017;35:311-315.

A competitive advantage by neonatally engrafted human glial progenitors yields mice whose brains are chimeric for human glia.

Neonatally transplanted human glial progenitor cells (hGPCs) densely engraft and myelinate the hypomyelinated shiverer mouse. 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 hypomyelinated shiverer brain 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 cells in vivo.

Modeling cognition and disease using human glial chimeric mice.

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 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.

CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance.

The naked mole rat (NMR) is the longest-lived rodent, resistant to multiple age-related diseases including neurodegeneration. However, the mechanisms underlying the NMR's resistance to neurodegenerative diseases remain elusive. Here, we isolated oligodendrocyte progenitor cells (OPCs) from NMRs and compared their transcriptome with that of other mammals. Extracellular matrix (ECM) genes best distinguish OPCs of long- and short-lived species. Notably, expression levels of CD44, an ECM-binding protein that has been suggested to contribute to NMR longevity by mediating the effect of hyaluronan (HA), are not only high in OPCs of long-lived species but also positively correlate with longevity in multiple cell types/tissues. We found that CD44 localizes to the endoplasmic reticulum (ER) and enhances basal ATF6 activity. CD44 modifies proteome and membrane properties of the ER and enhances ER stress resistance in a manner dependent on unfolded protein response regulators without the requirement of HA. HA-independent role of CD44 in proteostasis regulation may contribute to mammalian longevity.

Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain.

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.

Cell-intrinsic glial pathology is conserved across human and murine models of Huntington's disease.

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.

Stem cell-based strategies for treating pediatric disorders of myelin.

The pediatric leukodystrophies comprise a category of disease manifested by neonatal or childhood deficiencies in myelin production or maintenance; these may be due to hereditary defects in one or more genes critical to the initiation of myelination, as in Pelizaeus-Merzbacher Disease, or to enzymatic deficiencies with aberrant substrate accumulation-related dysfunction, as in the lysosomal storage disorders. Despite differences in both phenotype and natural history, these disorders are all essentially manifested by a profound deterioration in neurological function with age. A congenital deficit in forebrain myelination is also noted in children with the periventricular leukomalacia of cerebral palsy, another major source of neurological morbidity. In light of the wide range of disorders to which congenital hypomyelination and/or postnatal demyelination may contribute, and the relative homogeneity of central oligodendrocytes and their progenitors, the pediatric leukodystrophies may be especially attractive targets for cell-based therapeutic strategies. As a result, glial progenitor cells (GPCs), which can give rise to new myelinogenic oligodendrocytes, have become of great interest as potential therapeutic vectors for the restoration of myelin to the hypomyelinated or dysmyelinated childhood CNS. In addition, by distributing themselves throughout the deficient host neuraxis after perinatal allograft, and giving rise to astrocytes as well as oligodendrocytes, glial progenitors appear to be of potential great utility in rectifying enzymatic deficiencies. In this review, we focus on current efforts to develop the use of isolated human GPCs as transplantable agents both for mediating enzymatic restoration to the enzyme-deficient brain and for therapeutic myelination in the disorders of congenital hypomyelination.

Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain.

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.

Dysregulated Glial Differentiation in Schizophrenia May Be Relieved by Suppression of SMAD4- and REST-Dependent Signaling.

Astrocytic differentiation is developmentally impaired in patients with childhood-onset schizophrenia (SCZ). To determine why, we used genetic gain- and loss-of-function studies to establish the contributions of differentially expressed transcriptional regulators to the defective differentiation of glial progenitor cells (GPCs) produced from SCZ patient-derived induced pluripotent cells (iPSCs). Negative regulators of the bone morphogenetic protein (BMP) pathway were upregulated in SCZ GPCs, including BAMBI, FST, and GREM1, whose overexpression retained SCZ GPCs at the progenitor stage. SMAD4 knockdown (KD) suppressed the production of these BMP inhibitors by SCZ GPCs and rescued normal astrocytic differentiation. In addition, the BMP-regulated transcriptional repressor REST was upregulated in SCZ GPCs, and its KD similarly restored normal glial differentiation. REST KD also rescued potassium-transport-associated gene expression and K+ uptake, which were otherwise deficient in SCZ glia. These data suggest that the glial differentiation defect in childhood-onset SCZ, and its attendant disruption in K+ homeostasis, may be rescued by targeting BMP/SMAD4- and REST-dependent transcription.

Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation.

Huntington's disease (HD) is characterized by hypomyelination and neuronal loss. To assess the basis for myelin loss in HD, we generated bipotential glial progenitor cells (GPCs) from human embryonic stem cells (hESCs) derived from mutant Huntingtin (mHTT) embryos or normal controls and performed RNA sequencing (RNA-seq) to assess mHTT-dependent changes in gene expression. In human GPCs (hGPCs) derived from 3 mHTT hESC lines, transcription factors associated with glial differentiation and myelin synthesis were sharply downregulated relative to normal hESC GPCs; NKX2.2, OLIG2, SOX10, MYRF, and their downstream targets were all suppressed. Accordingly, when mHTT hGPCs were transplanted into hypomyelinated shiverer mice, the resultant glial chimeras were hypomyelinated; this defect could be rescued by forced expression of SOX10 and MYRF by mHTT hGPCs. The mHTT hGPCs also manifested impaired astrocytic differentiation and developed abnormal fiber architecture. White matter involution in HD is thus a product of the cell-autonomous, mHTT-dependent suppression of glial differentiation.

Human iPSC Glial Mouse Chimeras Reveal Glial Contributions to Schizophrenia.

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.

Human glia can both induce and rescue aspects of disease phenotype in Huntington disease.

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.

Human glial chimeric mice reveal astrocytic dependence of JC virus infection.

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.

Glial progenitor cell-based treatment and modeling of neurological disease.

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.

CD140a identifies a population of highly myelinogenic, migration-competent and efficiently engrafting human oligodendrocyte progenitor cells.

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.

Defective glial maturation in vanishing white matter disease.

Vanishing white matter (VWM) disease is a genetic leukoencephalopathy linked to mutations in the eukaryotic translation initiation factor 2B. It is a disease of infants, children, and adults who experience a slowly progressive neurologic deterioration with episodes of rapid clinical worsening triggered by stress and eventually leading to death. Characteristic neuropathologic findings include cystic degeneration of the white matter with scarce reactive gliosis, dysmorphic astrocytes, and paucity of myelin despite an increase in oligodendrocytic density. To assess whether a defective maturation of macroglia may be responsible for the feeble gliosis and lack of myelin, weinvestigated the maturation status of astrocytes and oligodendrocytes in the brains of 8 VWM patients, 4 patients with other white matter disorders and 6 age-matched controls with a combination of immunocytochemistry, histochemistry, scratch-wound assays, Western blot, and quantitative polymerase chain reaction. We observed increased proliferation and a defect in the maturation of VWM astrocytes. They show an anomalous composition of their intermediate filament network with predominance of the δ-isoform of the glial fibrillary acidic protein and an increase in the heat shock protein αB-crystallin, supporting the possibility that a deficiency in astrocyte function may contribute to the loss of white matter in VWM. We also demonstrated a significant increase in numbers of premyelinating oligodendrocyte progenitors in VWM, which may explain the coexistence of oligodendrocytosis and myelin paucity in the patients' white matter.

Fate determination of adult human glial progenitor cells.

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.

Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse.

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 x 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.