Publications by authors named "Steven L Stice"

89 Publications

Development of an objective index, neural activity score (NAS), reveals neural network ontogeny and treatment effects on microelectrode arrays.

Sci Rep 2021 Apr 27;11(1):9110. Epub 2021 Apr 27.

Regenerative Bioscience Center, University of Georgia, Athens, GA, USA.

Microelectrode arrays (MEAs) are valuable tools for electrophysiological analysis, providing assessment of neural network health and development. Analysis can be complex, however, requiring intensive processing of large data sets consisting of many activity parameters, leading to information loss as studies subjectively report relatively few metrics in the interest of simplicity. In screening assays, many groups report simple overall activity (i.e. firing rate) but omit network connectivity changes (e.g. burst characteristics and synchrony) that may not be evident from basic parameters. Our goal was to develop an objective process to capture most of the valuable information gained from MEAs in neural development and toxicity studies. We implemented principal component analysis (PCA) to reduce the high dimensionality of MEA data. Upon analysis, we found the first principal component was strongly correlated to time, representing neural culture development; therefore, factor loadings were used to create a single index score-named neural activity score (NAS)-reflecting neural maturation. For validation, we applied NAS to studies analyzing various treatments. In all cases, NAS accurately recapitulated expected results, suggesting viability of NAS to measure network health and development. This approach may be adopted by other researchers using MEAs to analyze complicated treatment effects and multicellular interactions.
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http://dx.doi.org/10.1038/s41598-021-88675-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079414PMC
April 2021

Exploring the predictive value of lesion topology on motor function outcomes in a porcine ischemic stroke model.

Sci Rep 2021 Feb 15;11(1):3814. Epub 2021 Feb 15.

Regenerative Bioscience Center, University of Georgia, Athens, GA, USA.

Harnessing the maximum diagnostic potential of magnetic resonance imaging (MRI) by including stroke lesion location in relation to specific structures that are associated with particular functions will likely increase the potential to predict functional deficit type, severity, and recovery in stroke patients. This exploratory study aims to identify key structures lesioned by a middle cerebral artery occlusion (MCAO) that impact stroke recovery and to strengthen the predictive capacity of neuroimaging techniques that characterize stroke outcomes in a translational porcine model. Clinically relevant MRI measures showed significant lesion volumes, midline shifts, and decreased white matter integrity post-MCAO. Using a pig brain atlas, damaged brain structures included the insular cortex, somatosensory cortices, temporal gyri, claustrum, and visual cortices, among others. MCAO resulted in severely impaired spatiotemporal gait parameters, decreased voluntary movement in open field testing, and higher modified Rankin Scale scores at acute timepoints. Pearson correlation analyses at acute timepoints between standard MRI metrics (e.g., lesion volume) and functional outcomes displayed moderate R values to functional gait outcomes. Moreover, Pearson correlation analyses showed higher R values between functional gait deficits and increased lesioning of structures associated with motor function, such as the putamen, globus pallidus, and primary somatosensory cortex. This correlation analysis approach helped identify neuroanatomical structures predictive of stroke outcomes and may lead to the translation of this topological analysis approach from preclinical stroke assessment to a clinical biomarker.
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http://dx.doi.org/10.1038/s41598-021-83432-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884696PMC
February 2021

Magnetic Resonance Imaging and Gait Analysis Indicate Similar Outcomes Between Yucatan and Landrace Porcine Ischemic Stroke Models.

Front Neurol 2020 21;11:594954. Epub 2021 Jan 21.

Regenerative Bioscience Center, University of Georgia, Athens, GA, United States.

The Stroke Therapy Academic Industry Roundtable (STAIR) has recommended that novel therapeutics be tested in a large animal model with similar anatomy and physiology to humans. The pig is an attractive model due to similarities in brain size, organization, and composition relative to humans. However, multiple pig breeds have been used to study ischemic stroke with potentially differing cerebral anatomy, architecture and, consequently, ischemic stroke pathologies. The objective of this study was to characterize brain anatomy and assess spatiotemporal gait parameters in Yucatan (YC) and Landrace (LR) pigs pre- and post-stroke using magnetic resonance imaging (MRI) and gait analysis, respectively. Ischemic stroke was induced via permanent middle cerebral artery occlusion (MCAO). MRI was performed pre-stroke and 1-day post-stroke. Structural and diffusion-tensor sequences were performed at both timepoints and analyzed for cerebral characteristics, lesion diffusivity, and white matter changes. Spatiotemporal and relative pressure gait measurements were collected pre- and 2-days post-stroke to characterize and compare acute functional deficits. The results from this study demonstrated that YC and LR pigs exhibit differences in gross brain anatomy and gait patterns pre-stroke with MRI and gait analysis showing statistical differences in the majority of parameters. However, stroke pathologies in YC and LR pigs were highly comparable post-stroke for most evaluated MRI parameters, including lesion volume and diffusivity, hemisphere swelling, ventricle compression, caudal transtentorial and foramen magnum herniation, showing no statistical difference between the breeds. In addition, post-stroke changes in velocity, cycle time, swing percent, cadence, and mean hoof pressure showed no statistical difference between the breeds. These results indicate significant differences between pig breeds in brain size, anatomy, and motor function pre-stroke, yet both demonstrate comparable brain pathophysiology and motor outcomes post-stroke. The conclusions of this study suggest pigs of these different breeds generally show a similar ischemic stroke response and findings can be compared across porcine stroke studies that use different breeds.
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http://dx.doi.org/10.3389/fneur.2020.594954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859633PMC
January 2021

Semi-Automated Cell and Tissue Analyses Reveal Regionally Specific Morphological Alterations of Immune and Neural Cells in a Porcine Middle Cerebral Artery Occlusion Model of Stroke.

Front Cell Neurosci 2020 22;14:600441. Epub 2021 Jan 22.

Regenerative Bioscience Center, University of Georgia, Athens, GA, United States.

Histopathological analysis of cellular changes in the stroked brain provides critical information pertaining to inflammation, cell death, glial scarring, and other dynamic injury and recovery responses. However, commonly used manual approaches are hindered by limitations in speed, accuracy, bias, and the breadth of morphological information that can be obtained. Here, a semi-automated high-content imaging (HCI) and CellProfiler histological analysis method was developed and used in a Yucatan miniature pig permanent middle cerebral artery occlusion (pMCAO) model of ischemic stroke to overcome these limitations. Evaluation of 19 morphological parameters in IBA1 microglia/macrophages, GFAP astrocytes, NeuN neuronal, FactorVIII vascular endothelial, and DCX neuroblast cell areas was conducted on porcine brain tissue 4 weeks post pMCAO. Out of 19 morphological parameters assessed in the stroke perilesional and ipsilateral hemisphere regions (38 parameters), a significant change in measured IBA1 parameters, GFAP parameters, NeuN parameters, FactorVIII parameters, and DCX parameters were observed in stroked vs. non-stroked animals. Principal component analysis (PCA) and correlation analyses demonstrated that stroke-induced significant and predictable morphological changes that demonstrated strong relationships between IBA1, GFAP, and NeuN areas. Ultimately, this unbiased, semi-automated HCI and CellProfiler histopathological analysis approach revealed regional and cell specific morphological signatures of immune and neural cells after stroke in a highly translational porcine model. These identified features can provide information of disease pathogenesis and evolution with high resolution, as well as be used in therapeutic screening applications.
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http://dx.doi.org/10.3389/fncel.2020.600441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862775PMC
January 2021

Electrophysiological Analysis of Brain Organoids: Current Approaches and Advancements.

Front Neurosci 2020 12;14:622137. Epub 2021 Jan 12.

Regenerative Bioscience Center, University of Georgia, Athens, GA, United States.

Brain organoids, or cerebral organoids, have become widely used to study the human brain . As pluripotent stem cell-derived structures capable of self-organization and recapitulation of physiological cell types and architecture, brain organoids bridge the gap between relatively simple two-dimensional human cell cultures and non-human animal models. This allows for high complexity and physiological relevance in a controlled setting, opening the door for a variety of applications including development and disease modeling and high-throughput screening. While technologies such as single cell sequencing have led to significant advances in brain organoid characterization and understanding, improved functional analysis (especially electrophysiology) is needed to realize the full potential of brain organoids. In this review, we highlight key technologies for brain organoid development and characterization, then discuss current electrophysiological methods for brain organoid analysis. While electrophysiological approaches have improved rapidly for two-dimensional cultures, only in the past several years have advances been made to overcome limitations posed by the three-dimensionality of brain organoids. Here, we review major advances in electrophysiological technologies and analytical methods with a focus on advances with applicability for brain organoid analysis.
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http://dx.doi.org/10.3389/fnins.2020.622137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835643PMC
January 2021

An integrative multivariate approach for predicting functional recovery using magnetic resonance imaging parameters in a translational pig ischemic stroke model.

Neural Regen Res 2021 May;16(5):842-850

Regenerative Bioscience Center; Neuroscience, Biomedical and Health Sciences Institute; Department of Animal and Dairy Science, College of Agricultural and Environmental Sciences, University of Georgia, Athens, GA, USA.

Magnetic resonance imaging (MRI) is a clinically relevant, real-time imaging modality that is frequently utilized to assess stroke type and severity. However, specific MRI biomarkers that can be used to predict long-term functional recovery are still a critical need. Consequently, the present study sought to examine the prognostic value of commonly utilized MRI parameters to predict functional outcomes in a porcine model of ischemic stroke. Stroke was induced via permanent middle cerebral artery occlusion. At 24 hours post-stroke, MRI analysis revealed focal ischemic lesions, decreased diffusivity, hemispheric swelling, and white matter degradation. Functional deficits including behavioral abnormalities in open field and novel object exploration as well as spatiotemporal gait impairments were observed at 4 weeks post-stroke. Gaussian graphical models identified specific MRI outputs and functional recovery variables, including white matter integrity and gait performance, that exhibited strong conditional dependencies. Canonical correlation analysis revealed a prognostic relationship between lesion volume and white matter integrity and novel object exploration and gait performance. Consequently, these analyses may also have the potential of predicting patient recovery at chronic time points as pigs and humans share many anatomical similarities (e.g., white matter composition) that have proven to be critical in ischemic stroke pathophysiology. The study was approved by the University of Georgia (UGA) Institutional Animal Care and Use Committee (IACUC; Protocol Number: A2014-07-021-Y3-A11 and 2018-01-029-Y1-A5) on November 22, 2017.
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http://dx.doi.org/10.4103/1673-5374.297079DOI Listing
May 2021

Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry.

Nanoscale Res Lett 2020 Aug 24;15(1):170. Epub 2020 Aug 24.

Regenerative Bioscience Center, University of Georgia, Athens, GA, 30602, USA.

Extracellular vesicles (EVs) are nanosized lipid bilayer-bound vesicles that are naturally secreted from most cell types as a communication mechanism to deliver proteins, lipids, and genetic material. Despite the therapeutic potential of EVs, there is limited information on EV uptake kinetics and specificity. Here, we optimized an imaging flow cytometry (IFC)-based platform to quantitatively assess dose, time, and recipient cell specificity effects on human embryonic kidney cell (HEK293T) EV internalization in a high-throughput manner. We found that HEK293T EV uptake is an active process that is dose and time dependent. Further, the selectivity of EV uptake was quantified in vitro, and we found that HEK293T EVs were internalized at higher quantities by cells of the same origin. Lastly, neural stem cells internalized significantly more HEK293T EVs relative to mature neurons, suggesting that stem cells or progenitors, which are more metabolically active than terminally differentiated cells, may have higher rates of active EV internalization. The characterization of EV uptake, notably specificity, dose and time dependence, and kinetic assays will help inform and develop targeted and efficient EV-based therapeutics.
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http://dx.doi.org/10.1186/s11671-020-03399-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445225PMC
August 2020

Tissue and Stem Cell Sourced Extracellular Vesicle Communications with Microglia.

Stem Cell Rev Rep 2021 Apr;17(2):357-368

Regenerative Bioscience Center, Department of Animal and Dairy Science, Rhodes Center for Animal and Dairy Science, University of Georgia, 425 River Road, Athens, GA, 30602, USA.

Extracellular vesicles (EVs), nano- to micro- sized vesicles released from cells, have garnered attention in recent years for their role in intercellular communication. Specifically, EVs from various cell sources including stem cells, have shown to have an exacerbatory or therapeutic effect in the content of pro- and anti-inflammatory environments through their interaction with immune recipient cells. This review aims to the coalescence information surrounding EVs derived from various sources and their interaction with microglia in neutral, anti, and pro- inflammatory environments. Overall, in homeostatic environments, EVs from many CNS lineages have been shown to have specific interactions with recipient microglia. In complex inflammatory environments, such as the tumor micro-environment (TME), EVs have been shown to further influence immune dampening through transition of microglia to a more M2-like phenotype. While not advantageous in the TME, this effect can be harnessed therapeutically in proinflammatory neurological conditions such as stroke, Alzheimer's, and Parkinson's. EVs derived from various stem cell and non-stem cell derived sources were found to attenuate proinflammatory responses in microglia in in vitro and in vivo models of these conditions. EVs loaded with anti-inflammatory therapeutics furthered this anti-inflammatory effect on recipient microglia. Graphical Abstract Extracellular Vesicles (EVs) from multiple cells types modulate microglial polarization. Cartoon depicting common ways microglia are activated through inflammatory and disease processes. EVs, derived from stem and non-stem sources, have been shown to attenuate proinflammatory responses in in vitro and in vivo.
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http://dx.doi.org/10.1007/s12015-020-10011-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036211PMC
April 2021

Development of 3D neuromuscular bioactuators.

APL Bioeng 2020 Mar 10;4(1):016107. Epub 2020 Mar 10.

Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

Neuronal control of skeletal muscle bioactuators represents a critical milestone toward the realization of future biohybrid machines that may generate complex motor patterns and autonomously navigate through their environment. Animals achieve these feats using neural networks that generate robust firing patterns and coordinate muscle activity through neuromuscular units. Here, we designed a versatile 3D neuron-muscle co-culture platform to serve as a test-bed for neuromuscular bioactuators. We used our platform in conjunction with microelectrode array electrophysiology to study the roles of synergistic interactions in the co-development of neural networks and muscle tissues. Our platform design enables co-culture of a neuronal cluster with up to four target muscle actuators, as well as quantification of muscle contraction forces. Using engineered muscle tissue targets, we first demonstrated the formation of functional neuromuscular bioactuators. We then investigated possible roles of long-range interactions in neuronal outgrowth patterns and observed preferential outgrowth toward muscles compared to the acellular matrix or fibroblasts, indicating muscle-specific chemotactic cues acting on motor neurons. Next, we showed that co-cultured muscle strips exhibited significantly higher spontaneous contractility as well as improved sarcomere assembly compared to muscles cultured alone. Finally, we performed microelectrode array measurements on neuronal cultures, which revealed that muscle-conditioned medium enhances overall neural firing rates and the emergence of synchronous bursting patterns. Overall, our study illustrates the significance of neuron-muscle cross talk for the development of neuromuscular bioactuators.
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http://dx.doi.org/10.1063/1.5134477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064368PMC
March 2020

--Labeled Cells Under the Tongue Epithelium Serve as Progenitors for Taste Bud Cells That Are Mainly Type III and Keratin 8-Low.

Stem Cells Dev 2020 05 24;29(10):638-647. Epub 2020 Mar 24.

Regenerative Bioscience Center, Department of Animal and Dairy Science, College of Agricultural and Environmental Sciences, University of Georgia, Athens, Georgia, USA.

Taste bud cells are specialized epithelial cells that undergo continuous turnover, and thus require active progenitors for their renewal and an intact taste function. Our previous studies suggested that a population of taste bud cells originates from outside of the surrounding tongue epithelium-previously regarded sole source of taste bud progenitors. In this study, we demonstrated that (SRY-related HMG-box gene 10)-expressing cells, known to be in the migrating neural crest, were also distributed in taste bud-surrounding tissue compartments under the tongue epithelium, that is, the connective tissue core of taste papillae and von Ebner's glands. By lineage tracing of -expressing cells using , a model driven by the endogenous promoter, crossing with a reporter line R26-tdTomato (tdT), we found -labeled tdT cells within taste buds in all three types of taste papillae (fungiform, circumvallate, and foliate) as well as in the soft palate in postnatal mice. The tdT taste bud cells were progressively more abundant along the developmental stages, from virtually zero at birth to over 35% in adults. Most of tdT taste bud cells had a low intensity of immunosignals of Keratin 8 (a widely used taste bud cell marker). In circumvallate taste buds, tdT signals were co-localized principally with a type III taste bud cell marker, less so with type I and II cell makers. Together, our data demonstrate a novel progenitor source for taste buds of postnatal mice--labeled cells in the connective tissue core and/or von Ebner's glands.
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http://dx.doi.org/10.1089/scd.2020.0022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232695PMC
May 2020

Neural Stem Cell Extracellular Vesicles Disrupt Midline Shift Predictive Outcomes in Porcine Ischemic Stroke Model.

Transl Stroke Res 2020 08 6;11(4):776-788. Epub 2019 Dec 6.

Regenerative Bioscience Center, University of Georgia, Athens, GA, 30602, USA.

Magnetic resonance imaging (MRI) is a clinically relevant non-invasive imaging tool commonly utilized to assess stroke progression in real time. This study investigated the utility of MRI as a predictive measure of clinical and functional outcomes when a stroke intervention is withheld or provided, in order to identify biomarkers for stroke functional outcome under these conditions. Fifteen MRI and ninety functional parameters were measured in a middle cerebral artery occlusion (MCAO) porcine ischemic stroke model. Multiparametric analysis of correlations between MRI measurements and functional outcome was conducted. Acute axial and coronal midline shift (MLS) at 24 h post-stroke were associated with decreased survival and recovery measured by modified Rankin scale (mRS) and were significantly correlated with 52 measured acute (day 1 post) and chronic (day 84 post) gait and behavior impairments in non-treated stroked animals. These results suggest that MLS may be an important non-invasive biomarker that can be used to predict patient outcomes and prognosis as well as guide therapeutic intervention and rehabilitation in non-treated animals and potentially human patients that do not receive interventional treatments. Neural stem cell-derived extracellular vesicle (NSC EV) was a disruptive therapy because NSC EV administration post-stroke disrupted MLS correlations observed in non-treated stroked animals. MLS was not associated with survival and functional outcomes in NSC EV-treated animals. In contrast to untreated animals, NSC EVs improved stroked animal outcomes regardless of MLS severity.
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http://dx.doi.org/10.1007/s12975-019-00753-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340639PMC
August 2020

Extracellular Vesicles Mediate Neuroprotection and Functional Recovery after Traumatic Brain Injury.

J Neurotrauma 2020 06 10;37(11):1358-1369. Epub 2020 Feb 10.

Regenerative Bioscience Center, University of Georgia, Athens, Georgia, USA.

The lack of effective therapies for moderate-to-severe traumatic brain injuries (TBIs) leaves patients with lifelong disabilities. Neural stem cells (NSCs) have demonstrated great promise for neural repair and regeneration. However, direct evidence to support their use as a cell replacement therapy for neural injuries is currently lacking. We hypothesized that NSC-derived extracellular vesicles (NSC EVs) mediate repair indirectly after TBI by enhancing neuroprotection and therapeutic efficacy of endogenous NSCs. We evaluated the short-term effects of acute intravenous injections of NSC EVs immediately following a rat TBI. Male NSC EV-treated rats demonstrated significantly reduced lesion sizes, enhanced presence of endogenous NSCs, and attenuated motor function impairments 4 weeks post-TBI, when compared with vehicle- and TBI-only male controls. Although statistically not significant, we observed a therapeutic effect of NSC EVs on brain lesion volume, nestin expression, and behavioral recovery in female subjects. Our study demonstrates the neuroprotective and functional benefits of NSC EVs for treating TBI and points to gender-dependent effects on treatment outcomes, which requires further investigation.
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http://dx.doi.org/10.1089/neu.2019.6443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249471PMC
June 2020

The Oxysterol 7-Ketocholesterol Reduces Zika Virus Titers in Vero Cells and Human Neurons.

Viruses 2018 12 30;11(1). Epub 2018 Dec 30.

Department of Infectious Diseases, Department of Population Health, Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.

Zika virus (ZIKV) is an emerging flavivirus responsible for a major epidemic in the Americas beginning in 2015. ZIKV associated with maternal infection can lead to neurological disorders in newborns, including microcephaly. Although there is an abundance of research examining the neurotropism of ZIKV, we still do not completely understand the mechanism by which ZIKV targets neural cells or how to limit neural cell infection. Recent research suggests that flaviviruses, including ZIKV, may hijack the cellular autophagy pathway to benefit their replication. Therefore, we hypothesized that ZIKV replication would be impacted when infected cells were treated with compounds that target the autophagy pathway. We screened a library of 94 compounds known to affect autophagy in both mammalian and insect cell lines. A subset of compounds that inhibited ZIKV replication without affecting cellular viability were tested for their ability to limit ZIKV replication in human neurons. From this second screen, we identified one compound, 7-ketocholesterol (7-KC), which inhibited ZIKV replication in neurons without significantly affecting neuron viability. Interestingly, 7-KC induces autophagy, which would be hypothesized to increase ZIKV replication, yet it decreased virus production. Time-of-addition experiments suggest 7-KC inhibits ZIKV replication late in the replication cycle. While 7-KC did not inhibit RNA replication, it decreased the number of particles in the supernatant and the relative infectivity of the released particles, suggesting it interferes with particle budding, release from the host cell, and particle integrity.
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http://dx.doi.org/10.3390/v11010020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356585PMC
December 2018

Strain-Dependent Consequences of Zika Virus Infection and Differential Impact on Neural Development.

Viruses 2018 10 9;10(10). Epub 2018 Oct 9.

Department of Infectious Diseases, Department of Population Health, Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.

Maternal infection with Zika virus (ZIKV) during pregnancy can result in neonatal abnormalities, including neurological dysfunction and microcephaly. Experimental models of congenital Zika syndrome identified neural progenitor cells as a target of viral infection. Neural progenitor cells are responsible for populating the developing central nervous system with neurons and glia. Neural progenitor dysfunction can lead to severe birth defects, namely, lissencephaly, microcephaly, and cognitive deficits. For this study, the consequences of ZIKV infection in human pluripotent stem cell-derived neural progenitor (hNP) cells and neurons were evaluated. ZIKV isolates from Asian and African lineages displayed lineage-specific replication kinetics, cytopathic effects, and impacts on hNP function and neuronal differentiation. The currently circulating ZIKV isolates exhibit a unique profile of virulence, cytopathic effect, and impaired cellular functions that likely contribute to the pathological mechanism of congenital Zika syndrome. The authors found that infection with Asian-lineage ZIKV isolates impaired the proliferation and migration of hNP cells, and neuron maturation. In contrast, the African-lineage infections resulted in abrupt and extensive cell death. This work furthers the understanding of ZIKV-induced brain pathology.
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http://dx.doi.org/10.3390/v10100550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212967PMC
October 2018

Chronic Electrical Stimulation Promotes the Excitability and Plasticity of ESC-derived Neurons following Glutamate-induced Inhibition In vitro.

Sci Rep 2018 Jul 19;8(1):10957. Epub 2018 Jul 19.

Regenerative Bioscience Center, ADS Complex, University of Georgia, Athens, Georgia.

Functional electrical stimulation (FES) is rapidly gaining traction as a therapeutic tool for mediating the repair and recovery of the injured central nervous system (CNS). However, the underlying mechanisms and impact of these stimulation paradigms at a molecular, cellular and network level remain largely unknown. In this study, we used embryonic stem cell (ESC)-derived neuron and glial co-cultures to investigate network maturation following acute administration of L-glutamate, which is a known mediator of excitotoxicity following CNS injury. We then modulated network maturation using chronic low frequency stimulation (LFS) and direct current stimulation (DCS) protocols. We demonstrated that L-glutamate impaired the rate of maturation of ESC-derived neurons and glia immediately and over a week following acute treatment. The administration of chronic LFS and DCS protocols individually following L-glutamate infusion significantly promoted the excitability of neurons as well as network synchrony, while the combination of LFS/DCS did not. qRT-PCR analysis revealed that LFS and DCS alone significantly up-regulated the expression of excitability and plasticity-related transcripts encoding N-methyl-D-aspartate (NMDA) receptor subunit (NR2A), brain-derived neurotrophic factor (BDNF) and Ras-related protein (RAB3A). In contrast, the simultaneous administration of LFS/DCS down-regulated BDNF and RAB3A expression. Our results demonstrate that LFS and DCS stimulation can modulate network maturation excitability and synchrony following the acute administration of an inhibitory dose of L-glutamate, and upregulate NR2A, BDNF and RAB3A gene expression. Our study also provides a novel framework for investigating the effects of electrical stimulation on neuronal responses and network formation and repair after traumatic brain injury.
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http://dx.doi.org/10.1038/s41598-018-29069-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053382PMC
July 2018

Human Neural Stem Cell Extracellular Vesicles Improve Recovery in a Porcine Model of Ischemic Stroke.

Stroke 2018 05 12;49(5):1248-1256. Epub 2018 Apr 12.

Regenerative Bioscience Center (R.L.W., E.E.K., B.J.J., S.S., F.D.W., S.L. Stice)

Background And Purpose: Recent work from our group suggests that human neural stem cell-derived extracellular vesicle (NSC EV) treatment improves both tissue and sensorimotor function in a preclinical thromboembolic mouse model of stroke. In this study, NSC EVs were evaluated in a pig ischemic stroke model, where clinically relevant end points were used to assess recovery in a more translational large animal model.

Methods: Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAO), and either NSC EV or PBS treatment was administered intravenously at 2, 14, and 24 hours post-MCAO. NSC EV effects on tissue level recovery were evaluated via magnetic resonance imaging at 1 and 84 days post-MCAO. Effects on functional recovery were also assessed through longitudinal behavior and gait analysis testing.

Results: NSC EV treatment was neuroprotective and led to significant improvements at the tissue and functional levels in stroked pigs. NSC EV treatment eliminated intracranial hemorrhage in ischemic lesions in NSC EV pigs (0 of 7) versus control pigs (7 of 8). NSC EV-treated pigs exhibited a significant decrease in cerebral lesion volume and decreased brain swelling relative to control pigs 1-day post-MCAO. NSC EVs significantly reduced edema in treated pigs relative to control pigs, as assessed by improved diffusivity through apparent diffusion coefficient maps. NSC EVs preserved white matter integrity with increased corpus callosum fractional anisotropy values 84 days post-MCAO. Behavior and mobility improvements paralleled structural changes as NSC EV-treated pigs exhibited improved outcomes, including increased exploratory behavior and faster restoration of spatiotemporal gait parameters.

Conclusions: This study demonstrated for the first time that in a large animal model novel NSC EVs significantly improved neural tissue preservation and functional levels post-MCAO, suggesting NSC EVs may be a paradigm changing stroke therapeutic.
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http://dx.doi.org/10.1161/STROKEAHA.117.020353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916046PMC
May 2018

Human Neural Stem Cell Extracellular Vesicles Improve Tissue and Functional Recovery in the Murine Thromboembolic Stroke Model.

Transl Stroke Res 2018 10 28;9(5):530-539. Epub 2017 Dec 28.

ArunA Biomedical, Athens, GA, 30602, USA.

Over 700 drugs have failed in stroke clinical trials, an unprecedented rate thought to be attributed in part to limited and isolated testing often solely in "young" rodent models and focusing on a single secondary injury mechanism. Here, extracellular vesicles (EVs), nanometer-sized cell signaling particles, were tested in a mouse thromboembolic (TE) stroke model. Neural stem cell (NSC) and mesenchymal stem cell (MSC) EVs derived from the same pluripotent stem cell (PSC) line were evaluated for changes in infarct volume as well as sensorimotor function. NSC EVs improved cellular, tissue, and functional outcomes in middle-aged rodents, whereas MSC EVs were less effective. Acute differences in lesion volume following NSC EV treatment were corroborated by MRI in 18-month-old aged rodents. NSC EV treatment has a positive effect on motor function in the aged rodent as indicated by beam walk, instances of foot faults, and strength evaluated by hanging wire test. Increased time with a novel object also indicated that NSC EVs improved episodic memory formation in the rodent. The therapeutic effect of NSC EVs appears to be mediated by altering the systemic immune response. These data strongly support further preclinical development of a NSC EV-based stroke therapy and warrant their testing in combination with FDA-approved stroke therapies.
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http://dx.doi.org/10.1007/s12975-017-0599-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6132936PMC
October 2018

Zika Virus Exhibits Lineage-Specific Phenotypes in Cell Culture, in Aedes aegypti Mosquitoes, and in an Embryo Model.

Viruses 2017 12 16;9(12). Epub 2017 Dec 16.

Department of Infectious Diseases, Department of Population Health, Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.

Zika virus (ZIKV) has quietly circulated in Africa and Southeast Asia for the past 65 years. However, the recent ZIKV epidemic in the Americas propelled this mosquito-borne virus to the forefront of flavivirus research. Based on historical evidence, ZIKV infections in Africa were sporadic and caused mild symptoms such as fever, skin rash, and general malaise. In contrast, recent Asian-lineage ZIKV infections in the Pacific Islands and the Americas are linked to birth defects and neurological disorders. The aim of this study is to compare replication, pathogenicity, and transmission efficiency of two historic and two contemporary ZIKV isolates in cell culture, the mosquito host, and an embryo model to determine if genetic variation between the African and Asian lineages results in phenotypic differences. While all tested isolates replicated at similar rates in Vero cells, the African isolates displayed more rapid viral replication in the mosquito C6/36 cell line, yet they exhibited poor infection rates in mosquitoes compared to the contemporary Asian-lineage isolates. All isolates could infect chicken embryos; however, infection with African isolates resulted in higher embryo mortality than infection with Asian-lineage isolates. These results suggest that genetic variation between ZIKV isolates can significantly alter experimental outcomes.
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http://dx.doi.org/10.3390/v9120383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744157PMC
December 2017

The African Zika virus MR-766 is more virulent and causes more severe brain damage than current Asian lineage and dengue virus.

Development 2017 11 9;144(22):4114-4124. Epub 2017 Oct 9.

Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA 90033, USA

The Zika virus (ZIKV) has two lineages, Asian and African, and their impact on developing brains has not been compared. Dengue virus (DENV) is a close family member of ZIKV and co-circulates with ZIKV. Here, we performed intracerebral inoculation of embryonic mouse brains with dengue virus 2 (DENV2), and found that DENV2 is sufficient to cause smaller brain size due to increased cell death in neural progenitor cells (NPCs) and neurons. Compared with the currently circulating Asian lineage of ZIKV (MEX1-44), DENV2 grows slower, causes less neuronal death and fails to cause postnatal animal death. Surprisingly, our side-by-side comparison uncovered that the African ZIKV isolate (MR-766) is more potent at causing brain damage and postnatal lethality than MEX1-44. In comparison with MEX1-44, MR-766 grows faster in NPCs and in the developing brain, and causes more pronounced cell death in NPCs and neurons, resulting in more severe neuronal loss. Together, these results reveal that DENV2 is sufficient to cause smaller brain sizes, and suggest that the ZIKV African lineage is more toxic and causes more potent brain damage than the Asian lineage.
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http://dx.doi.org/10.1242/dev.156752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719247PMC
November 2017

Determination of chlorpyrifos and its metabolites in cells and culture media by liquid chromatography-electrospray ionization tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2017 Sep 18;1063:112-117. Epub 2017 Aug 18.

Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, GA 30602-2352, United States. Electronic address:

A sensitive method to simultaneously quantitate chlorpyrifos, chlorpyrifos oxon and the detoxified product 3,5,6-trichloro-2-pyridinol (TCP) was developed using either liquid-liquid extraction for culture media samples, or protein precipitation for cell samples. Multiple reaction monitoring in positive ion mode was applied for the detection of chlorpyrifos and chlorpyrifos oxon, and selected ion recording in negative mode was applied to detect TCP. The method provided linear ranges from 5 to 500, 0.2-20 and 20-2000ng/mL for media samples and from 0.5-50, 0.02-2 and 2-200ng/million cells for CPF, CPO and TCP, respectively. The method was validated using selectivity, linearity, precision, accuracy, recovery, stability and dilution tests. All relative standard deviations (RSDs) and relative errors (REs) for QC samples were within 15% (except for LLOQ, within 20%). This method has been successfully applied to study the neurotoxicity and metabolism of chlorpyrifos in a human neuronal model.
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http://dx.doi.org/10.1016/j.jchromb.2017.08.010DOI Listing
September 2017

Tracking and Quantification of Magnetically Labeled Stem Cells using Magnetic Resonance Imaging.

Adv Funct Mater 2016 Jun 17;26(22):3899-3915. Epub 2016 Feb 17.

Bioimaging Research Center, Regenerative Bioscience Center, and Department of Physics University of Georgia, Athens, GA. 30602, USA.

Stem cell based therapies have critical impacts on treatments and cures of diseases such as neurodegenerative or cardiovascular disease. In vivo tracking of stem cells labeled with magnetic contrast agents is of particular interest and importance as it allows for monitoring of the cells' bio-distribution, viability, and physiological responses. Herein, recent advances are introduced in tracking and quantification of super-paramagnetic iron oxide (SPIO) nanoparticles-labeled cells with magnetic resonance imaging, a noninvasive approach that can longitudinally monitor transplanted cells. This is followed by recent translational research on human stem cells that are dual-labeled with green fluorescence protein (GFP) and SPIO nanoparticles, then transplanted and tracked in a chicken embryo model. Cell labeling efficiency, viability, and cell differentiation are also presented.
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http://dx.doi.org/10.1002/adfm.201504444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526633PMC
June 2016

From the Cover: AstrocytesAre Protective Against Chlorpyrifos Developmental Neurotoxicity in Human Pluripotent Stem Cell-Derived Astrocyte-Neuron Cocultures.

Toxicol Sci 2017 06;157(2):410-420

Department of Animal and Dairy Science, Interdisciplinary Toxicology Program, University of Georgia, Athens, GA, USA.

Human neural progenitor cells are capable of independent, directed differentiation into astrocytes, oligodendrocytes and neurons and thus offer a potential cell source for developmental neurotoxicity (DNT) systems. Human neural progenitor-derived astrocyte-neuron cocultured at defined ratios mimic cellular heterogeneity and interaction in the central nervous system. Cytochrome P450 enzymes are expressed at a relatively high level in astrocytes and may play a critical role in the biotransformation of endogenous or exogenous compounds, including chlorpyrifos, an organophosphate insecticide that affects the central nervous system. P450 enzymes metabolize chlorpyrifos to chlorpyrifos-oxon, which is then metabolized primarily to 3, 5, 6-trichloropyridinol in addition to diethylphosphate and diethylthiophosphate. These end metabolites are less neurotoxic than chlorpyrifos and chlorpyrifos-oxon. Our objective was to identify the interactive role of astrocytes and neurons in chlorpyrifos-induced human DNT. In neuron-only cultures, chlorpyrifos inhibited neurite length, neurite number and branch points per neuron in a dose-dependent manner during a 48 h exposure, starting at 10 μM. However, in astrocyte-neuron cocultures, astrocytes protected neurons from the effects of chlorpyrifos at higher concentrations, up to and including 30 μM chlorpyrifos and endogenous astrocyte P450 enzymes effectively metabolized chlorpyrifos. The P450 inhibitor SKF525A partly negated the protective effect of astrocytes, allowing reduction in branch points with chlorpyrifos (10 μM). Thus, the scalable and defined astrocyte-neuron cocultures model that we established here has potentially identified a role for P450 enzymes in astrocytic neuroprotection against chlorpyrifos and provides a novel model for addressing DNT in a more accurate multicellular environment.
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http://dx.doi.org/10.1093/toxsci/kfx056DOI Listing
June 2017

Pig Induced Pluripotent Stem Cell-Derived Neural Rosettes Parallel Human Differentiation Into Sensory Neural Subtypes.

Cell Reprogram 2017 04 7;19(2):88-94. Epub 2017 Mar 7.

1 Regenerative Bioscience Center, University of Georgia , Rhodes Center for Animal and Dairy Science, Athens, Georgia .

The pig is the large animal model of choice for study of nerve regeneration and wound repair. Availability of porcine sensory neural cells would conceptually allow for analogous cell-based peripheral nerve regeneration in porcine injuries of similar severity and size to those found in humans. After recently reporting that porcine (or pig) induced pluripotent stem cells (piPSCs) differentiate into neural rosette (NR) structures similar to human NRs, here we demonstrate that pig NR cells could differentiate into neural crest cells and other peripheral nervous system-relevant cell types. Treatment with either bone morphogenetic protein 4 or fetal bovine serum led to differentiation into BRN3A-positive sensory cells and increased expression of sensory neuron TRK receptor gene family: TRKA, TRKB, and TRKC. Porcine sensory neural cells would allow determination of parallels between human and porcine cells in response to noxious stimuli, analgesics, and reparative mechanisms. In vitro differentiation of pig sensory neurons provides a novel model system for neural cell subtype specification and would provide a novel platform for the study of regenerative therapeutics by elucidating the requirements for innervation following injury and axonal survival.
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http://dx.doi.org/10.1089/cell.2016.0057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016089PMC
April 2017

Derivation of chicken induced pluripotent stem cells tolerant to Newcastle disease virus-induced lysis through multiple rounds of infection.

Virol J 2016 12 5;13(1):205. Epub 2016 Dec 5.

US National Poultry Research Center, Exotic and Emerging Avian Viral Diseases Research Unit, Southeast Poultry Research Laboratory, Athens, GA, 30605, USA.

Background: Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a devastating disease of poultry and wild birds. ND is prevented by rigorous biocontainment and vaccination. One potential approach to prevent spread of the virus is production of birds that show innate resistance to NDV-caused disease. Induced pluripotent stem cell (iPSC) technology allows adult cells to be reprogrammed into an embryonic stem cell-like state capable of contributing to live offspring and passing on unique traits in a number of species. Recently, iPSC approaches have been successfully applied to avian cells. If chicken induced pluripotent stem cells (ciPSCs) are genetically or epigenetically modified to resist NDV infection, it may be possible to generate ND resistant poultry. There is limited information on the potential of ciPSCs to be infected by NDV, or the capacity of these cells to become resistant to infection. The aim of the present work was to assess the characteristics of the interaction between NDV and ciPSCs, and to develop a selection method that would increase tolerance of these cells to NDV-induced cellular damage.

Results: Results showed that ciPSCs were permissive to infection with NDV, and susceptible to virus-mediated cell death. Since ciPSCs that survived infection demonstrated the ability to recover quickly, we devised a system to select surviving cells through multiple infection rounds with NDV. ciPSCs that sustained 9 consecutive infections had a statistically significant increase in survival (up to 36 times) compared to never-infected ciPSCs upon NDV infection (tolerant cells). Increased survival was not caused by a loss of permissiveness to NDV replication. RNA sequencing followed by enrichment pathway analysis showed that numerous metabolic pathways where differentially regulated between tolerant and never-infected ciPSCs.

Conclusions: Results demonstrate that ciPSCs are permissive to NDV infection and become increasingly tolerant to NDV under selective pressure, indicating that this system could be applied to study mechanisms of cellular tolerance to NDV.
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http://dx.doi.org/10.1186/s12985-016-0659-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5139146PMC
December 2016

High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure.

BMC Pharmacol Toxicol 2016 12 1;17(1):62. Epub 2016 Dec 1.

Interdisciplinary Toxicology Program, University of Georgia, Athens, GA, 30602, USA.

Background: Our objective was to test neural active compounds in a human developmental neurotoxicity (DNT) model that represents neural tube stages of vulnerability. Previously we showed that 14 days in vitro (DIV 14) was sufficient to generate cryopreserved neuronal cells for post thaw neurite recovery assays. However, short exposure and assessment may not detect toxicants that affect an early neurogenesis continuum, from a mitotic human neural progenitor (hNP) cell population through the course of neurite outgrowth in differentiating neurons. Therefore, we continuously exposed differentiating hNP cells from DIV 0 through DIV 14 to known toxicants and endocrine active compounds in order to assess at DIV 14 effects of these compounds in a human DNT maturation model for neurogenesis.

Methods: The Human DNT continuum (DIV 0 to DIV 14) was determined using immunocytochemistry for SOX1+ (proliferating hNP) and HuC/D+ (post mitotic neurons). The cumulative effects of five compounds was observed on neurite outgrowth in (βIII-tubulin+) and (HuC/D+) cells using high content imaging. All data were analyzed using a one-way ANOVA with a significance threshold of p < 0.05.

Results: During maturation in vitro, the neural cultures transitioned from uniform hNP cells (DIV 0) to predominantly mature post mitotic neuronal neurons (HuC/D+, 65%; DIV14) but also maintained a smaller population of hNP cells (SOX1+). Using this DNT maturation model system, Bis-1, testosterone, and β-estradiol inhibited neuronal maturation at micromolar levels but were unaffected by acetaminophen. β-estradiol also disrupted neurite extension at 10 μM. Treating cells in this window with Bisphenol A (BPA) significantly inhibited neurite outgrowth and branching in these continuum cultures but only at the highest concentrations tested (10 μM).

Conclusions: Cumulative effects of neurotoxicant exposure during a maturation continuum altered human neurogenesis at lower exposure levels than observed in acute exposure of static cryopreserved neurite recovery neurons cultures. Unlike prior acute studies, β-estradiol was highly toxic when present throughout the continuum and cytotoxicity was manifested starting early in the continuum via a non-estrogen receptor α (ER α) mechanism. Therefore, the effect of neural developmental neurotoxins can and should be determined during the dynamic process of human neural maturation.
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http://dx.doi.org/10.1186/s40360-016-0107-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131404PMC
December 2016

Labeling and analysis of chicken taste buds using molecular markers in oral epithelial sheets.

Sci Rep 2016 11 17;6:37247. Epub 2016 Nov 17.

Regenerative Bioscience Center, Department of Animal and Dairy Science, College of Agricultural and Environmental Sciences, University of Georgia, Athens, GA, USA.

In chickens, the sensory organs for taste are the taste buds in the oral cavity, of which there are ~240-360 in total number as estimated by scanning electron microscopy (SEM). There is not an easy way to visualize all taste buds in chickens. Here, we report a highly efficient method for labeling chicken taste buds in oral epithelial sheets using the molecular markers Vimentin and α-Gustducin. Immediate tissue fixation following incubation with sub-epithelially injected proteases enabled us to peel off whole epithelial sheets, leaving the shape and integrity of the tissue intact. In the peeled epithelial sheets, taste buds labeled with antibodies against Vimentin and α-Gustducin were easily identified and counted under a light microscope and many more taste buds, patterned in rosette-like clusters, were found than previously reported with SEM. Broiler-type, female-line males have more taste buds than other groups and continue to increase the number of taste buds over stages after hatch. In addition to ovoid-shaped taste buds, big tube-shaped taste buds were observed in the chicken using 2-photon microscopy. Our protocol for labeling taste buds with molecular markers will factilitate future mechanistic studies on the development of chicken taste buds in association with their feeding behaviors.
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http://dx.doi.org/10.1038/srep37247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5112566PMC
November 2016

Zika Virus Induced Mortality and Microcephaly in Chicken Embryos.

Stem Cells Dev 2016 11 17;25(22):1691-1697. Epub 2016 Oct 17.

5 Department of Animal and Dairy Science, Regenerative Bioscience Center, College of Agriculture and Environmental Science, University of Georgia , Athens, Georgia .

The explosive spread of the Zika virus (ZIKV) through South and Central America has been linked to an increase in congenital birth defects, specifically microcephaly. Representative rodent models for investigating infections include direct central nervous system (CNS) injections late in pregnancy and transplacental transmission in immunodeficient mice. Microcephaly in humans may be the result of infection occurring early in pregnancy, therefore recapitulating that the human course of ZIKV infection should include normal embryo exposed to ZIKV during the first trimester. In ovo development of the chicken embryo closely mirrors human fetal neurodevelopment and, as a comparative model, could provide key insights into both temporal and pathophysiological effects of ZIKV. Chick embryos were directly infected early and throughout incubation with ZIKV isolated from a Mexican mosquito in January 2016. High doses of virus caused embryonic lethality. In a subset of lower dosed embryos, replicating ZIKV was present in various organs, including the CNS, throughout development. Surviving ZIKV-infected embryos presented a microcephaly-like phenotype. Chick embryos were longitudinally monitored by magnetic resonance imaging that documented CNS structural malformations, including enlarged ventricles (30% increase) and stunted cortical growth (decreased telencephalon by 18%, brain stem by 32%, and total brain volume by 18%), on both embryonic day 15 (E15) and E20 of development. ZIKV-induced microcephaly was observed with inoculations of as few as 2-20 viral particles. The chick embryo model presented ZIKV embryonic lethal effects and progressive CNS damage similar to microcephaly.
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http://dx.doi.org/10.1089/scd.2016.0231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453490PMC
November 2016

Generation of Chimeras from Porcine Induced Pluripotent Stem Cells.

Methods Mol Biol 2015 ;1330:153-67

Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, 425 River Road, RM 316, Athens, GA, 30602, USA.

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g., gene knock-in, knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs, microinjection of piPSCs into embryos, embryo transfer and production of chimeric animals based on successful protocols.
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http://dx.doi.org/10.1007/978-1-4939-2848-4_14DOI Listing
September 2016

Generation of Avian Induced Pluripotent Stem Cells.

Methods Mol Biol 2015 ;1330:89-99

Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, 425 River Road, RM 316, Athens, GA, 30602, USA.

Avian species are among the most diverse vertebrates on our planet and significantly contribute to the balance of the ecology. They are also important food source and serve as a central animal model to decipher developmental biology and disease principles. Derivation of induced pluripotent stem cells (iPSCs) from avian species would enable conservation of genetic diversity as well as offer a valuable cell source that facilitates the use of avian models in many areas of basic and applied research. In this chapter, we describe methods used to successfully reprogram quail fibroblasts into iPSCs by using human transcription factors and the techniques critical to the characterization of their pluripotency.
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http://dx.doi.org/10.1007/978-1-4939-2848-4_9DOI Listing
September 2016

Development, characterization and optimization of a new suspension chicken-induced pluripotent cell line for the production of Newcastle disease vaccine.

Biologicals 2016 Jan 14;44(1):24-32. Epub 2015 Nov 14.

Exotic and Emerging Avian Viral Diseases Research Unit, Southeast Poultry Research Laboratory, Athens, GA 30605, USA. Electronic address:

Traditionally, substrates for production of viral poultry vaccines have been embryonated eggs or adherent primary cell cultures. The difficulties and cost involved in scaling up these substrates in cases of increased demand have been a limitation for vaccine production. Here, we assess the ability of a newly developed chicken-induced pluripotent cell line, BA3, to support replication and growth of Newcastle disease virus (NDV) LaSota vaccine strain. The characteristics and growth profile of the cells were also investigated. BA3 cells could grow in suspension in different media to a high density of up to 7.0 × 10(6) cells/mL and showed rapid proliferation with doubling time of 21 h. Upon infection, a high virus titer of 1.02 × 10(8) EID50/mL was obtained at 24 h post infection using a multiplicity of infection (MOI) of 5. In addition, the cell line was shown to be free of endogenous and exogenous Avian Leukosis viruses, Reticuloendotheliosis virus, Fowl Adenovirus, Marek's disease virus, and several Mycoplasma species. In conclusion, BA3 cell line is potentially an excellent candidate for vaccine production due to its highly desirable industrially friendly characteristics of growing to high cell density and capability of growth in serum free medium.
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http://dx.doi.org/10.1016/j.biologicals.2015.09.002DOI Listing
January 2016