Sergei Grigoryan

Bar Ilan University, Gan, Tel Aviv, Israel

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Publications (3)12.58 Total impact

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    ABSTRACT: Retrograde axonal transport of the neurotropic alphaherpesvirus Varicella zoster virus (VZV) from vesicles at the skin results in sensory neuron infection and establishment of latency. Reactivation from latency leads to painful herpes zoster. The lack of a suitable animal model of these processes for the highly human-restricted VZV has resulted in a dearth of knowledge regarding the axonal transport of VZV. We recently demonstrated VZV infection of distal axons, leading to subsequent capsid transport to the neuronal somata, and replication and release of infectious virus using a new model based on neurons derived from human embryonic stem cells (hESC). In the present study, we perform a kinetic analysis of the retrograde transport of green fluorescent protein-tagged ORF23 in VZV capsids using hESC-derived neurons compartmentalized microfluidic chambers and time-lapse video microscopy. The motion of the VZV was discontinuous, showing net retrograde movement with numerous short pauses and reversals in direction. Velocities measured were higher 1 h after infection than 6 h after infection, while run lengths were similar at both time points. The hESC-derived neuron model was also used to show that reduced neuronal spread by a VZV loss-of-function mutant for ORF7 is not due to the prevention of axonal infection and transport of the virus to the neuronal somata. hESC-derived neurons are, therefore, a powerful model for studying axonal transport of VZV and molecular characteristics of neuronal infection.
    Journal of NeuroVirology 08/2012; 18(6). DOI:10.1007/s13365-012-0124-z · 2.85 Impact Factor
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    ABSTRACT: Study of the human neurotrophic herpesvirus varicella-zoster virus (VZV) and of its ability to infect neurons has been severely limited by strict viral human tropism and limited availability of human neurons for experimentation. Human embryonic stem cells (hESC) can be differentiated to all the cell types of the body including neurons and are therefore a potentially unlimited source of human neurons to study their interactions with human neurotropic viruses. We report here reproducible infection of hESC-derived neurons by cell-associated green fluorescent protein (GFP)-expressing VZV. hESC-derived neurons expressed GFP within 2 days after incubation with mitotically inhibited MeWo cells infected with recombinant VZV expressing GFP as GFP fusions to VZV proteins or under an independent promoter. VZV infection was confirmed by immunostaining for immediate-early and viral capsid proteins. Infection of hESC-derived neurons was productive, resulting in release into the medium of infectious virions that appeared fully assembled when observed by electron microscopy. We also demonstrated, for the first time, VZV infection of axons and retrograde transport from axons to neuronal cell bodies using compartmented microfluidic chambers. The use of hESC-derived human neurons in conjunction with fluorescently tagged VZV shows great promise for the study of VZV neuronal infection and axonal transport and has potential for the establishment of a model for VZV latency in human neurons.
    Journal of Virology 07/2011; 85(13):6220-33. DOI:10.1128/JVI.02396-10 · 4.65 Impact Factor
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    ABSTRACT: Schwann cells (SC), the glial cells of peripheral nerves, are involved in many diseases including Charcot Marie Tooth and neurofibromatosis, and play a pivotal role in peripheral nerve regeneration. Although it is possible to obtain human SC from nerve biopsies, they are difficult to maintain and expand in culture. Here we describe an efficient system for directing the differentiation of human embryonic stem cells (hESC) into cells with the morphological and molecular characteristics of SC. Neurospheres were generated from hESC using stromal cell induction and grown under conditions supportive of SC differentiation. After 8 weeks, hESC-derived SC expressed characteristic markers GFAP, S100, HNK1, P75, MBP and PMP-22, and were observed in close association with hESC-derived neurites. ~60% of the cells were double-immunostained for the SC markers GFAP/S100. RT-PCR analysis confirmed the expression of GFAP, S100, P75, PMP-22 and MBP and demonstrated expression of the SC markers P0, KROX20 and PLP in the cultures. Expression of CAD19 was observed in 2 and 4 week cultures and then was down-regulated, consistent with its expression in SC precursor, but not mature stages. Co-culture of hESC-derived SC with rat, chick or hESC-derived axons in compartmentalized microfluidic chambers resulted in tight association of the SC with axons. Apparent wrapping of the axons by SC was occasionally observed, suggestive of myelination. Our method for generating SC from hESC makes available a virtually unlimited source of human SC for studies of their role in nerve regeneration and modeling of disease.
    Stem cell reviews 10/2010; 7(2):394-403. DOI:10.1007/s12015-010-9198-2 · 5.08 Impact Factor