A human neuron injury model for molecular studies of axonal regeneration

Ziegler, Lina; Segal-Ruder, Yael; Coppola, Giovanni; Reis, Arbel; Geschwind, Daniel H.; Fainzilber, Mike; Goldstein, Ronald S.

doi:10.1016/j.expneurol.2009.09.019

Cited by 12 publications

(9 citation statements)

References 37 publications

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“…Taken together with our published observations (14), the results presented above suggest that hESC cells are not infected by VZV, while neurons differentiated from them are infected by and replicate and release VZV. The method used in our laboratory for generating neurons from hESC involves (i) induction by coculture with the murine stromal cell line PA6, (ii) mechanical dissection of specific colonies from the stromal cells and culture in suspension resulting in the generation of neurospheres, and (iii) plating the neurospheres in medium lacking mitogens but containing neuronal survival factors (14,18,30). We therefore tested hESC-derived neural progenitors at each of these stages for their ability to be infected by VZV.…”

Section: Resultsmentioning

confidence: 99%

Varicella-Zoster Virus Infects Human Embryonic Stem Cell-Derived Neurons and Neurospheres but Not Pluripotent Embryonic Stem Cells or Early Progenitors

Dukhovny

Sloutskin

Markus

et al. 2012

J Virol

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bPluripotent human stem cells are a powerful tool for the generation of differentiated cells that can be used for the study of human disease. We recently demonstrated that neurons derived from pluripotent human embryonic stem cells (hESC) can be infected by the highly host-restricted human alphaherpesvirus varicella-zoster virus (VZV), permitting the interaction of VZV with neurons to be readily evaluated in culture. In the present study, we examine whether pluripotent hESC and neural progenitors at intermediate stages of differentiation are permissive for VZV infection. We demonstrate here that VZV infection is blocked in naïve hESC. A block to VZV replication is also seen when a bacterial artificial chromosome (BAC) containing the VZV genome is transfected into hESC. In contrast, related alphaherpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PrV) productively infect naïve hESC in a cell-free manner, and PrV replicates from a BAC transfected into hESC. Neurons differentiate from hESC via neural progenitor intermediates, as is the case in the embryo. The first in vitro stage at which permissiveness of hESC-derived neural precursors to VZV replication is observed is upon formation of "neurospheres," immediately after detachment from the inductive stromal feeder layer. These findings suggest that hESC may be useful in deciphering the yet enigmatic mechanisms of specificity of VZV infection and replication. V aricella-zoster virus (VZV) replication is highly host restricted, growing efficiently only in human cells. In varicella, VZV typically infects and replicates in cutaneous fibroblasts and epidermal cells as well as several types of immune cells. VZV infections of central nervous system (CNS) vasculature are also not uncommonly observed, the virus infecting smooth muscle actinexpressing cells in vessel walls (16). VZV infects effectively primarily in a cell-associated manner in vitro, and it is thought that cell-to-cell spread occurs in most tissues. Cell-free virus is made in vivo by keratinocytes and is present in cutaneous vesicles (8), and released VZV appears to be an important component of T cell-toskin transmission in vivo (reviewed in reference 1).VZV infection of neurons is essential for establishment of latency and the ability to reactivate to cause herpes zoster. Initial neuronal infection by VZV is via cutaneous axons and retrograde transport to peripheral somatic and autonomic ganglia and/or by infected circulating lymphocytes that infiltrate the ganglia (28). VZV replicates in both neurons and ganglionic support cells of somatic and cranial peripheral sensory ganglia both upon initial infection and upon reactivation. Importantly, VZV causes a plethora of CNS diseases (due at least in part to infection of the vasculature) (16) and ocular diseases (reviewed in reference 9). The growth of VZV in neurons and the interactions that govern latency and reactivation have proven to be difficult to study outside the human host because of the species restriction of infection and the limited...

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Section: Resultsmentioning

confidence: 99%

Varicella-Zoster Virus Infects Human Embryonic Stem Cell-Derived Neurons and Neurospheres but Not Pluripotent Embryonic Stem Cells or Early Progenitors

Dukhovny

Sloutskin

Markus

et al. 2012

J Virol

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“…The medium was changed every 3 days. Under these conditions, the cultures contain approximately 95% ␤III-tubulin-positive neurons, few glial fibrillary acidic proteinpositive (GFAP ϩ ) glial cells, and few Ki67-positive (Ki67 ϩ ) proliferating cells after 1 month of differentiation (33). The specific phenotypes of the neurons have not been determined, but approximately 10% express Brn3a/peripherin, a combination of markers characteristic of primary somatic sensory neurons of the DRG and trigeminal ganglion (19).…”

Section: Vol 85 2011 Vzv Infection Of Hesc-derived Neurons 6221mentioning

confidence: 99%

“…hESC differentiation into neurons has proven particularly useful in such studies since human neurons are difficult to obtain from biopsy specimen material. For example, hESCderived neurons were recently utilized for the study of molecular changes in regenerating human neurons after injury (33).…”

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confidence: 99%

Varicella-Zoster Virus (VZV) Infection of Neurons Derived from Human Embryonic Stem Cells: Direct Demonstration of Axonal Infection, Transport of VZV, and Productive Neuronal Infection

Markus

Grigoryan

Sloutskin

et al. 2011

J Virol

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106

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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.The interactions of the human neurotrophic herpesvirus varicella-zoster virus (VZV) with neurons have proven difficult to study because the virus shows fairly strict human specificity, and small-animal models do not fully recapitulate human disease. In humans, primary VZV infection follows viral inhalation and subsequent systemic delivery to the deep dermis of the skin via hemopoietic cells. In the course of the resulting disease (chickenpox), VZV infects sensory and sympathetic ganglion neurons, where it establishes a long period of latency. The infection of neurons may take place in the ganglia by circulating VZV-infected lymphocytes, or by virus infecting cutaneous nerve endings being retrogradely transported in the axon to the neuronal somata, as is the case with herpes simplex virus (HSV). VZV reactivation often leads to herpes zoster (shingles), a disease that is frequently associated with severe, debilitating, and often long-lasting intractable pain (postherpetic neuralgia) that is more often than not refractory to therapy.Few model systems of neuronal VZV infection have been developed. Two in vitro models are VZV infection of dissociated human neurons and intact human fetal dorsal root ganglia (DRG) (8, 9, 10). These studies have shed some light on VZV-neuronal interactions, demonstrating, for example, that VZV exerts antiapoptotic activities in neurons in the short term (maximum, 5 days) and that, unlike infected fibroblasts, infectious VZV is released from neurons.A human fetal DRG-SCID mouse model (22, 29; reviewed in reference 30) has al...

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“…Impact- and/or acceleration-induced brain traumatic injuries have already been the focus of many cellular and macroscopical studies through in vivo [3, 4, 5, 6, 7, 8, 9, 10], ex vivo [11, 12], in vitro [13, 14, 15, 16, 17], medical postanalysis [18, 19, 20] or modeling approaches [21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34]. However the specific effects of a blast—a pressure wave of finite amplitude generated by a rapid release of energy [35]—on the brain is still widely unkown.…”

Section: Introductionmentioning

confidence: 99%

Continuum modeling of a neuronal cell under blast loading

Jérusalem

Dao

2012

Acta Biomaterialia

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Traumatic brain injuries have recently been put under the spotlight as one of the most important causes of accidental brain dysfunctions. Significant experimental and modeling efforts are thus ongoing to study the associated biological, mechanical and physical mechanisms. In the field of cell mechanics, progresses are also being made at the experimental and modeling levels to better characterize many of the cell functions such as differentiation, growth, migration and death, among others. The work presented here aims at bridging both efforts by proposing a continuum model of neuronal cell submitted to blast loading. In this approach, cytoplasm, nucleus and membrane (plus cortex) are differentiated in a representative cell geometry, and different material constitutive models are adequately chosen for each one. The material parameters are calibrated against published experimental work of cell nanoindentation at multiple rates. The final cell model is ultimately subjected to blast loading within a complete fluid-structure interaction computational framework. The results are compared to the nanoindentation simulation and the specific effects of the blast wave on the pressure and shear levels at the interfaces are identified. As a conclusion, the presented model successfully captures some of the intrinsic intracellular phenomena occurring during its deformation under blast loading and potentially leading to cell damage. It suggests more particularly the localization of damage at the nucleus membrane similarly to what has already been observed at the overall cell membrane. This degree of damage is additionally predicted to be worsened by a longer blast positive phase duration. As a conclusion, the proposed model ultimately provides a new three dimensional computational tool to evaluate intracellular damage during blast loading.

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A human neuron injury model for molecular studies of axonal regeneration

Cited by 12 publications

References 37 publications

Varicella-Zoster Virus Infects Human Embryonic Stem Cell-Derived Neurons and Neurospheres but Not Pluripotent Embryonic Stem Cells or Early Progenitors

Varicella-Zoster Virus Infects Human Embryonic Stem Cell-Derived Neurons and Neurospheres but Not Pluripotent Embryonic Stem Cells or Early Progenitors

Varicella-Zoster Virus (VZV) Infection of Neurons Derived from Human Embryonic Stem Cells: Direct Demonstration of Axonal Infection, Transport of VZV, and Productive Neuronal Infection

Continuum modeling of a neuronal cell under blast loading

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