Imaging the dynamics of murine uterine contractions in early pregnancy†.

Uterine muscle contractility is essential for reproductive processes including sperm and embryo transport, and during the uterine cycle to remove menstrual effluent. Even still, uterine contractions have primarily been studied in the context of preterm labor. This is partly due to a lack of methods for studying the uterine muscle contractility in the intact organ. Here, we describe an imaging-based method to evaluate mouse uterine contractility of both the longitudinal and circular muscles in the cycling stages and in early pregnancy. By transforming the image-based data into three-dimensional spatiotemporal contractility maps, we calculate waveform characteristics of muscle contractions, including amplitude, frequency, wavelength, and velocity. We report that the native organ is highly contractile during the progesterone-dominant diestrus stage of the cycle when compared to the estrogen-dominant proestrus and estrus stages. We also observed that during the first phase of uterine embryo movement when clustered embryos move toward the middle of the uterine horn, contractions are dynamic and non-uniform between different segments of the uterine horn. In the second phase of embryo movement, contractions are more uniform and rhythmic throughout the uterine horn. Finally, in Lpar3-/- uteri, which display faster embryo movement, we observe global and regional increases in contractility. Our method provides a means to understand the wave characteristics of uterine smooth muscle in response to modulators and in genetic mutants. Better understanding uterine contractility in the early pregnancy stages is critical for the advancement of artificial reproductive technologies and a possibility of modulating embryo movement during clinical embryo transfers.

Glial progenitor cells of the adult human white and grey matter are contextually distinct.

Genomic analyses have revealed heterogeneity among glial progenitor cells (GPCs), but the compartment selectivity of human GPCs (hGPCs) is unclear. Here, we asked if GPCs of human grey and white brain matter are distinct in their architecture and associated gene expression. RNA profiling of NG2-defined hGPCs derived from adult human neocortex and white matter differed in their expression of genes involved in Wnt, NOTCH, BMP and TGFß signaling, suggesting compartment-selective biases in fate and self-renewal. White matter hGPCs over-expressed the BMP antagonists BAMBI and CHRDL1, suggesting their tonic suppression of astrocytic fate relative to cortical hGPCs, whose relative enrichment of cytoskeletal genes presaged their greater morphological complexity. In human glial chimeric mice, cortical hGPCs assumed larger and more complex morphologies than white matter hGPCs, and both were more complex than their mouse counterparts. These findings suggest that human grey and white matter GPCs comprise context-specific pools with distinct functional biases.

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.

Human Glial Chimeric Mice to Define the Role of Glial Pathology in Human Disease.

Human glial progenitor cells (hGPCs) can engraft, expand, and differentiate into functional oligodendrocytes and astrocytes when transplanted neonatally into murine hosts, in which they outcompete the host glial pool to ultimately colonize and dominate the recipient brains. When congenitally hypomyelinated mutants are used as hosts, the donor hGPCs generate myelinogenic oligodendrocytes as well as astrocytes, so that the recipient mice develop a largely humanized white matter, with entirely human-derived myelin. In addition, by neonatally engrafting hGPCs derived from patient- and disease-specific pluripotent stem cells, glial chimeric mice may be produced in which large proportions of all macroglial cells are not only human but also patient and disease specific. Human glial chimeric mice thus provide intriguing preparations by which to investigate the species-specific contributions of human glia to both cognition and human-selective neurodegenerative and neuropsychiatric diseases, as well as the potential for therapeutic glial cell replacement in these disorders. This review presents an overview of the uses, characteristics, and limitations of the human glial chimeric brain model, while providing a step-by-step protocol for the establishment of these mice.

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 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.