Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse.
ABSTRACT: 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.
Project description: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.
Project description: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.
Project description:The transcriptional program that controls oligodendrocyte maturation and central nervous system (CNS) myelination has not been fully characterized. In this study, we use high-throughput RNA sequencing to analyze how the loss of a key transcription factor, zinc finger protein 191 (ZFP191), results in oligodendrocyte development abnormalities and CNS hypomyelination. Using a previously described mutant mouse that is deficient in ZFP191 protein expression (Zfp191(null)), we demonstrate that key transcripts are reduced in the whole brain as well as within oligodendrocyte lineage cells cultured in vitro To determine whether the loss of myelin seen in Zfp191(null) mice contributes indirectly to these perturbations, we also examined the transcriptome of a well-characterized mouse model of hypomyelination, in which the myelin structural protein myelin basic protein (MBP) is deficient. Interestingly, Mbp(shi) (shiverer) mice had far fewer transcripts perturbed with the loss of myelin alone. This study demonstrates that the loss of ZFP191 disrupts expression of genes involved in oligodendrocyte maturation and myelination, largely independent from the loss of myelin. Nevertheless, hypomyelination in both mouse mutants results in the perturbation of lipid synthesis pathways, suggesting that oligodendrocytes have a feedback system that allows them to regulate myelin lipid synthesis depending on their myelinating state. The data presented are of potential clinical relevance as the human orthologs of the Zfp191 and MBP genes reside on a region of Chromosome 18 that is deleted in childhood leukodystrophies.
Project description:Neonatal engraftment by oligodendrocyte progenitor cells (OPCs) permits the myelination of the congenitally dysmyelinated brain. To establish a potential autologous source of these cells, we developed a strategy by which to differentiate human induced pluripotent stem cells (hiPSCs) into OPCs. From three hiPSC lines, as well as from human embryonic stem cells (hESCs), we generated highly enriched OLIG2(+)/PDGFR?(+)/NKX2.2(+)/SOX10(+) human OPCs, which could be further purified using fluorescence-activated cell sorting. hiPSC OPCs efficiently differentiated into both myelinogenic oligodendrocytes and astrocytes, in vitro and in vivo. Neonatally engrafted hiPSC OPCs robustly myelinated the brains of myelin-deficient shiverer mice and substantially increased their survival. The speed and efficiency of myelination by hiPSC OPCs was higher than that previously observed using fetal-tissue-derived OPCs, and no tumors from these grafts were noted as long as 9 months after transplant. These results suggest the potential utility of hiPSC-derived OPCs in treating disorders of myelin loss.
Project description:Shiverer-immunodeficient (Shi-id) mice demonstrate defective myelination in the central nervous system (CNS) and significant ataxia by 2 to 3 weeks of life. Expanded, banked human neural stem cells (HuCNS-SCs) were transplanted into three sites in the brains of neonatal or juvenile Shi-id mice, which were asymptomatic or showed advanced hypomyelination, respectively. In both groups of mice, HuCNS-SCs engrafted and underwent preferential differentiation into oligodendrocytes. These oligodendrocytes generated compact myelin with normalized nodal organization, ultrastructure, and axon conduction velocities. Myelination was equivalent in neonatal and juvenile mice by quantitative histopathology and high-field ex vivo magnetic resonance imaging, which, through fractional anisotropy, revealed CNS myelination 5 to 7 weeks after HuCNS-SC transplantation. Transplanted HuCNS-SCs generated functional myelin in the CNS, even in animals with severe symptomatic hypomyelination, suggesting that this strategy may be useful for treating dysmyelinating diseases.
Project description:440-kD ankyrinB is an alternatively spliced variant of 220-kD ankyrinB, with a predicted 220-kD sequence inserted between the membrane/spectrin binding domains and COOH-terminal domain (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 236:1372-1379). This paper presents the sequence of 2085 amino acids comprising the alternatively spliced portion of 440-kD ankyrinB, and provides evidence that much of the inserted sequence has the configuration of an extended random coil. Notable features of the inserted sequence include a hydrophilicity profile that contains few hydrophobic regions, and 220 predicted sites for phosphorylation by protein kinases (casein kinase 2, protein kinase C, and proline-directed protein kinase). Secondary structure and folding of the inserted amino acid residues were deduced from properties of recombinant polypeptides. Frictional ratios of 1.9-2.4 were calculated from Stokes radii and sedimentation coefficients, for polypeptides comprising 70% of the inserted sequence, indicating a highly asymmetric shape. Circular dichroism spectra of these polypeptides indicate a nonglobular structure with negligible alpha-helix or beta sheet folding. These results suggest a ball-and-chain model for 440-kD ankyrinB with a membrane-associated globular head domain and an extended filamentous tail domain encoded by the inserted sequence. Immunofluorescence and immunoblot studies of developing neonatal rat optic nerve indicate that 440-kD ankyrinB is selectively targeted to premyelinated axons, and that 440-kD ankyrinB disappears from these axons coincident with myelination. Hypomyelinated nerve tracts of the myelin-deficient Shiverer mice exhibit elevated levels of 440-kD ankyrinB. 440-kD ankyrinB thus is a specific component of unmyelinated axons and expression of 440-kD ankyrinB may be downregulated as a consequence of myelination.
Project description:The initiation of axoglial contact is considered a prerequisite for myelination, yet the role cell adhesion molecules (CAMs) play in mediating such interactions remains unclear. To examine the function of axoglial CAMs, we tested whether enhanced CAM-mediated adhesion between OLs and neurons could affect myelination. Here we show that increased expression of a membrane-bound extracellular domain of Cadm4 (Cadm4dCT) in cultured oligodendrocytes results in the production of numerous axoglial contact sites that fail to elongate and generate mature myelin. Transgenic mice expressing Cadm4dCT were hypomyelinated and exhibit multiple myelin abnormalities, including myelination of neuronal somata. These abnormalities depend on specific neuron-glial interaction as they were not observed when these OLs were cultured alone, on nanofibers, or on neurons isolated from mice lacking the axonal receptors of Cadm4. Our results demonstrate that tightly regulated axon-glia adhesion is essential for proper myelin targeting and subsequent membrane wrapping and lateral extension.
Project description:Neuroimaging classification procedures between normal and pathological subjects are sparse and highly dependent of an expert's clinical criterion. Here, we aimed to investigate whether possible brain structural network differences in the shiverer mouse mutant, a relevant animal model of myelin related diseases, can reflect intrinsic individual brain properties that allow the automatic discrimination between the shiverer and normal subjects. Common structural networks properties between shiverer (C3Fe.SWV Mbp(shi)/Mbp(shi), n?=?6) and background control (C3HeB.FeJ, n?=?6) mice are estimated and compared by means of three diffusion weighted MRI (DW-MRI) fiber tractography algorithms and a graph framework. Firstly, we found that brain networks of control group are significantly more clustered, modularized, efficient and optimized than those of the shiverer group, which presented significantly increased characteristic path length. These results are in line with previous structural/functional complex brain networks analysis that have revealed topologic differences and brain network randomization associated to specific states of human brain pathology. In addition, by means of network measures spatial representations and discrimination analysis, we show that it is possible to classify with high accuracy to which group each subject belongs, providing also a probability value of being a normal or shiverer subject as an individual anatomical classifier. The obtained correct predictions (e.g., around 91.6-100%) and clear spatial subdivisions between control and shiverer mice, suggest that there might exist specific network subspaces corresponding to specific brain disorders, supporting also the point of view that complex brain network analyses constitutes promising tools in the future creation of interpretable imaging biomarkers.
Project description:A quantitative magnetization transfer (qMT) technique was employed to quantify the ratio of the sizes of the bound and free water proton pools in ex vivo mouse brains. The goal was to determine the pool size ratio sensitivity to myelin. Fixed brains from both shiverer mice and control littermates were imaged. The pool size ratio in the corpus callosum of shiverer mice was substantially lower than that in the control mice, while there was no distinguishable difference in the pool size ratio in the gray matter. These results correlate with diffusion tensor imaging (DTI) derived radial diffusivity which previously was shown to reflect myelin integrity in this animal model. Histological study reveals the presence of myelin in control mice white matter and the absence of myelin in shiverer mice white matter, supporting the qMT and DTI results. Our findings support the view that qMT may be used for estimating myelin integrity.
Project description: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)?, 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.