Neural precursor cells derived from induced pluripotent stem cells exhibit reduced susceptibility to infection with a neurotropic coronavirus.

The present study examines the susceptibility of mouse induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) to infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Similar to NPCs derived from striatum of day 1 postnatal GFP-transgenic mice (GFP-NPCs), iPSC-derived NPCs (iPSC-NPCs) are able to differentiate into terminal neural cell types and express MHC class I and II in response to IFN-γ treatment. However, in contrast to postnatally-derived NPCs, iPSC-NPCs express low levels of carcinoembryonic antigen-cell adhesion molecule 1a (CEACAM1a), the surface receptor for JHMV, and are less susceptible to infection and virus-induced cytopathic effects. The relevance of this in terms of therapeutic application of NPCs resistant to viral infection is discussed

Doctor of Philosophy

dissertationNeural precursor cell (NPC) transplantation has emerged as a therapeutic option to treat several neurological disorders. Intracranial infection of mice with the JHM strain of mouse hepatitis virus (JHMV) results in a chronic demyelinating disease and thus has been used as a model to study multiple sclerosis (MS). The cause of MS remains unknown but viruses have been attributed to trigger the disease in genetically susceptible individuals, making it imperative to study the remyelination potential of NPCs in a virally induced demyelinating disease. We have previously shown that engraftment of fetal derived GFP-NPCs in spinal cords of JHMV-infected mice with established demyelination results in remyelination and axonal sparing. We have previously demonstrated that transplanted GFP-NPCs are susceptible to JHMV-induced cell death. From a clinical standpoint, donor-specific induced pluripotent stem cells (iPSC)-derived NPCs may be preferable to avoid the use of immunosuppressive drugs. Therefore, we sought to investigate whether iPSC derived NPCs are functionally similar to fetal-derived NPCs and whether they are susceptible to JHMV infection in a preclinical setting. iPSCNPCs are similar to GFP-NPCs in functionality as they are able to differentiate into oligodendrocytes, astrocytes, and neurons. However, iPSC-NPCs express low levels of the viral receptor CEACAM1a, making them resistant to JHMV infection and viralinduced cytopathic effects. Activated microglia can prevent maturation of oligodendrocyte precursor cells (OPCs) to myelinating oligodendrocytes and promote oxidative damage by releasing nitric oxide. In the presence of environmental insults, activated microglia can release IL- 6, IL-23, IFN-γ and TNF-α, all of which can be neurotoxic and neuroinflammatory. Microglia can also contribute to adaptive immunity by presenting antigen to CD4+ and CD8+ T cells that have entered the central nervous system (CNS) in response to infection. In order to determine the contribution of microglia to disease progression in the JHMV model, we used colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia in CNS of JHMV-infected mice. Microglia depletion results in increased viral titers, morbidity and, mortality and also modulates the immunological landscape of the CNS of JHMV-infected mice

VIRUS INDUCED FORMATION OF EXTRACELLULAR MICROVESICLES during COXSACKIEVIRUS DISSEMINATION IN THE HOST

Includes bibliographical references (pages 27-29).Coxsackievirus (CVB) is a relatively common enterovirus belonging to the\ud picornaviridae family that is capable of causing aseptic meningitis, pancreatitis and myocarditis in humans. Coxsackievirus B3 (CVB3) preferentially targets neural progenitor and stem cells (NPSCs) with lasting consequences in the central nervous system (CNS). We\ud genetically engineered a recombinant coxsackievirus expressing "fluorescent timer" protein (Timer-CVB3)."Fluorescent timer" protein undergoes a slow conversion from green to red over time, and thus, Timer-CVB3 can be utilized to detect the progression of virus infection. Upon infection with Timer-CVB3, partially differentiated neural progenitor and stem cells (NPSCs) and C2C12 myoblast cells released extracellular microvesicles (EMVs) containing matured "fluorescent timer" protein and infectious virus. Purified EMVs isolated from the supernatants of Timer-CVB3 infected cells were positive for the exosomal marker - flotillin-1, the autophagosomal marker - LC3-II, and viral capsid protein - VP-1. EMVs isolated by iodixanol isopycnic density purification were found in low density fractions consistent with membrane association distinct from high density viral fractions representing infectious virions. Detection of the lipidated form of LC3 protein (LC3-II) in EMVs observed within infected cell culture supernatants suggests involvement of the autophagy pathway during the release of EMVs. Also, viral protein co-localized with LC3 protein within EMVs identified in infected cultured cells, indicating that autophagy is an important process involved in release of shed microvesicles harboring infectious CVB3. Clarifying the role of these\ud infectious EMVs is important for understanding virus immune evasion and for the development of new antiviral therapies and vaccines

Neural precursor cells derived from induced pluripotent stem cells exhibit reduced susceptibility to infection with a neurotropic coronavirus.

The present study examines the susceptibility of mouse induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) to infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Similar to NPCs derived from striatum of day 1 postnatal GFP-transgenic mice (GFP-NPCs), iPSC-derived NPCs (iPSC-NPCs) are able to differentiate into terminal neural cell types and express MHC class I and II in response to IFN-γ treatment. However, in contrast to postnatally-derived NPCs, iPSC-NPCs express low levels of carcinoembryonic antigen-cell adhesion molecule 1a (CEACAM1a), the surface receptor for JHMV, and are less susceptible to infection and virus-induced cytopathic effects. The relevance of this in terms of therapeutic application of NPCs resistant to viral infection is discussed

Promoting remyelination through cell transplantation therapies in a model of viral-induced neurodegenerative disease.

Multiple sclerosis (MS) is a central nervous system (CNS) disease characterized by chronic neuroinflammation, demyelination, and axonal damage. Infiltration of activated lymphocytes and myeloid cells are thought to be primarily responsible for white matter damage and axonopathy. Several United States Food and Drug Administration-approved therapies exist that impede activated lymphocytes from entering the CNS thereby limiting new lesion formation in patients with relapse-remitting forms of MS. However, a significant challenge within the field of MS research is to develop effective and sustained therapies that allow for axonal protection and remyelination. In recent years, there has been increasing evidence that some kinds of stem cells and their derivatives seem to be able to mute neuroinflammation as well as promote remyelination and axonal integrity. Intracranial infection of mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in immune-mediated demyelination and axonopathy, making this an excellent model to interrogate the therapeutic potential of stem cell derivatives in evoking remyelination. This review provides a succinct overview of our recent findings using intraspinal injection of mouse CNS neural progenitor cells and human neural precursors into JHMV-infected mice. JHMV-infected mice receiving these cells display extensive remyelination associated with axonal sparing. In addition, we discuss possible mechanisms associated with sustained clinical recovery. Developmental Dynamics 248:43-52, 2019. © 2018 Wiley Periodicals, Inc

Microglia influence immune responses and restrict neurologic disease in response to central nervous system infection by a neurotropic murine coronavirus.

Intracranial (i.c.) inoculation of susceptible mice with a glial-tropic strain of mouse hepatitis virus (JHMV), a murine coronavirus, results in an acute encephalomyelitis followed by viral persistence in white matter tracts accompanied by chronic neuroinflammation and demyelination. Microglia serve numerous functions including maintenance of the healthy central nervous system (CNS) and are among the first responders to injury or infection. More recently, studies have demonstrated that microglia aid in tailoring innate and adaptive immune responses following infection by neurotropic viruses including flaviviruses, herpesviruses, and picornaviruses. These findings have emphasized an important role for microglia in host defense against these viral pathogens. In addition, microglia are also critical in optimizing immune-mediated control of JHMV replication within the CNS while restricting the severity of demyelination and enhancing remyelination. This review will highlight our current understanding of the molecular and cellular mechanisms by which microglia aid in host defense, limit neurologic disease, and promote repair following CNS infection by a neurotropic murine coronavirus