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Methods Mol Biol ; 1919: 175-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30656629


Limited access to primary tissue from various nonhuman primate (NHP) species represents a significant unmet need that hampers progress in understanding unique cellular diversity and gene regulation of specific tissues and organs in stem cell translational research. Most comparative biology studies have been limited to using postmortem tissue usually frozen specimens with limited utility for research. The generation of induced pluripotent stem cell (iPSC) lines from somatic cells, such as adult skin or blood cells, offers an alternative to invasive and ethically controversial interventions for acquiring tissue. Pluripotent iPSCs have virtually an unlimited capacity to proliferate and differentiate into all cell types of the body. We are generating high-quality validated NHP iPSC lines to offer to scientific community and facilitate their research programs. We use the non-integrative episomal vector system to generate iPSCs from NHP skin biopsies. In this chapter we describe the validation of NHP iPSC lines by confirming pluripotency and their propensity to differentiate into all three germ layers ectoderm, mesoderm, and endoderm according to established standards and measurable limits for a set of marker genes incorporated into a scorecard.

Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Medicina Regenerativa , Transcriptoma , Animais , Biópsia , Callithrix , Linhagem Celular , Corpos Embrioides/metabolismo , Perfilação da Expressão Gênica/métodos , Pele/citologia , Pele/metabolismo , Fluxo de Trabalho
Epilepsy Behav ; 89: 84-88, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30388666


OBJECTIVE: There is a high cost associated with recording quality video and electroencephalography (EEG) data in National Association of Epilepsy Center (NAEC) level IV epilepsy monitoring units (EMU). This study considers potential quality measures in EMUs for generalized tonic-clonic (GTC) seizures: types of safety signals, response time, and visibility of patient's limbs for semiology. These quality measures have been summarized across 12 EMUs to estimate response times to GTC seizures and the quality of video data that is captured during admissions. METHODS: Video electroencephalographies (vEEGs) from two prospective regulatory studies for the Brain Sentinel device were reviewed. A total of 232 subjects with a history of GTC seizures underwent routine clinical EMU stays. Fifty-four of the study subjects had 96 GTC seizures. The vEEG of events were reviewed for safety signal used, response time, and visibility of patient's limbs. RESULTS: The average response time from members of the hospital team was 22 s from electrographic generalization (minimum -37 s, maximum 111 s, two no response). For caregivers, average response was 11 s (minimum -15 s, maximum 33 s, 45 not present/no response). In 73% of events, the patient visibility was limited at seizure onset. In 55% of events with limited limb visibility, the visibility was improved (by removing sheets or improving camera angle) >30 s after start of the event. The primary safety signals were as follows: an alert from outside the patient room (54%), button press (23%), hospital team present at seizure start (14%), caregiver vocal alert (6%), and no response (2%). SIGNIFICANCE: The average response time of caregivers was twice as fast as the hospital team, underscoring the importance of having a person in the room during onset of a GTC seizure. Diagnostic yield could be improved with more timely removal of patient coverings. It was observed that when patients experienced a GTC seizure, 40% were fully or partially obscured for more than 30 s during the event, compromising the ability of epileptologists to evaluate semiology during seizure onset. Automated seizure alarms may help staff get to patients more quickly and improve diagnostic characterization.

Cuidadores/estatística & dados numéricos , Eletroencefalografia/normas , Epilepsia Tônico-Clônica/diagnóstico , Unidades Hospitalares/estatística & dados numéricos , Monitorização Fisiológica/estatística & dados numéricos , Segurança do Paciente/normas , Convulsões/diagnóstico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Eletroencefalografia/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica/normas , Estudos Prospectivos , Fatores de Tempo , Gravação em Vídeo , Adulto Jovem
Int J Mol Sci ; 19(9)2018 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-30227600


Humans and nonhuman primates (NHP) are similar in behavior and in physiology, specifically the structure, function, and complexity of the immune system. Thus, NHP models are desirable for pathophysiology and pharmacology/toxicology studies. Furthermore, NHP-derived induced pluripotent stem cells (iPSCs) may enable transformative developmental, translational, or evolutionary studies in a field of inquiry currently hampered by the limited availability of research specimens. NHP-iPSCs may address specific questions that can be studied back and forth between in vitro cellular assays and in vivo experimentations, an investigational process that in most cases cannot be performed on humans because of safety and ethical issues. The use of NHP model systems and cell specific in vitro models is evolving with iPSC-based three-dimensional (3D) cell culture systems and organoids, which may offer reliable in vitro models and reduce the number of animals used in experimental research. IPSCs have the potential to give rise to defined cell types of any organ of the body. However, standards for deriving defined and validated NHP iPSCs are missing. Standards for deriving high-quality iPSC cell lines promote rigorous and replicable scientific research and likewise, validated cell lines reduce variability and discrepancies in results between laboratories. We have derived and validated NHP iPSC lines by confirming their pluripotency and propensity to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) according to standards and measurable limits for a set of marker genes. The iPSC lines were characterized for their potential to generate neural stem cells and to differentiate into dopaminergic neurons. These iPSC lines are available to the scientific community. NHP-iPSCs fulfill a unique niche in comparative genomics to understand gene regulatory principles underlying emergence of human traits, in infectious disease pathogenesis, in vaccine development, and in immunological barriers in regenerative medicine.

Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Neurogênese , Animais , Callithrix , Técnicas de Cultura de Células , Linhagem da Célula , Células Cultivadas , Técnicas de Reprogramação Celular , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Células-Tronco Neurais/metabolismo , Pele/citologia
Euro Surveill ; 23(12)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29589577


BackgroundPrevious studies showed low levels of circulating hepatitis E virus (HEV) in Scotland. We aimed to reassess current Scottish HEV epidemiology. Methods: Blood donor samples from five Scottish blood centres, the minipools for routine HEV screening and liver transplant recipients were tested for HEV antibodies and RNA to determine seroprevalence and viraemia. Blood donor data were compared with results from previous studies covering 2004-08. Notified laboratory-confirmed hepatitis E cases (2009-16) were extracted from national surveillance data. Viraemic samples from blood donors (2016) and chronic hepatitis E transplant patients (2014-16) were sequenced. Results: Anti-HEV IgG seroprevalence varied geographically and was highest in Edinburgh where it increased from 4.5% in 2004-08) to 9.3% in 2014-15 (p = 0.001). It was most marked in donors < 35 years. HEV RNA was found in 1:2,481 donors, compared with 1:14,520 in 2011. Notified laboratory-confirmed cases increased by a factor of 15 between 2011 and 2016, from 13 to 206. In 2011-13, 1 of 329 transplant recipients tested positive for acute HEV, compared with six cases of chronic infection during 2014-16. Of 10 sequenced viraemic donors eight and all six patients were infected with genotype 3 clade 1 virus, common in European pigs. Conclusions: The seroprevalence, number of viraemic donors and numbers of notified laboratory-confirmed cases of HEV in Scotland have all recently increased. The causes of this change are unknown, but need further investigation. Clinicians in Scotland, particularly those caring for immunocompromised patients, should have a low threshold for testing for HEV.

Doadores de Sangue , Vírus da Hepatite E/isolamento & purificação , Hepatite E/epidemiologia , Hepatite E/virologia , Imunoglobulina G/sangue , RNA Viral/sangue , Viremia/virologia , Adolescente , Adulto , Feminino , Genótipo , Anticorpos Anti-Hepatite/sangue , Hepatite E/sangue , Hepatite E/transmissão , Vírus da Hepatite E/genética , Vírus da Hepatite E/imunologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Filogenia , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Escócia/epidemiologia , Estudos Soroepidemiológicos , Viremia/epidemiologia , Adulto Jovem
Stem Cells Transl Med ; 6(3): 877-885, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28297573


Optimal stem cell delivery procedures are critical to the success of the cell therapy approach. Variables such as flow rate, suspension solution, needle diameter, cell density, and tissue mechanics affect tissue penetration, backflow along the needle, and the dispersion and survival of injected cells during delivery. Most cell transplantation centers engaged in human clinical trials use custom-designed cannula needles, syringes, or catheters, sometimes precluding the use of magnetic resonance imaging (MRI)-guided delivery to target tissue. As a result, stem cell therapies may be hampered because more than 80% of grafted cells do not survive the delivery-for example, to the heart, liver/pancreas, and brain-which translates to poor patient outcomes. We developed a minimally invasive interventional MRI (iMRI) approach for intraoperatively imaging neural stem cell (NSC) delivery procedures. We used NSCs prelabeled with a contrast agent and real-time magnetic resonance imaging to guide the injection cannula to the target and to track the delivery of the cells into the putamen of baboons. We provide evidence that cell injection into the brain parenchyma follows a novel pulsatile mode of cellular discharge from the delivery catheter despite a constant infusion flow rate. The rate of cell infusion significantly affects the dispersion and viability of grafted cells. We report on our investigational use of a frameless navigation system for image-guided NSC transplantation using a straight cannula. Through submillimeter accuracy and real-time imaging, iMRI approaches may improve the safety and efficacy of neural cell transplantation therapies. Stem Cells Translational Medicine 2017;6:877-885.

Gânglios da Base/citologia , Imagem por Ressonância Magnética , Células-Tronco Neurais/transplante , Transplante de Células-Tronco/métodos , Animais , Sobrevivência Celular , Sistemas de Computação , Dextranos/química , Humanos , Nanopartículas de Magnetita/química , Células-Tronco Neurais/citologia , Papio , Imagens de Fantasmas
Brain Imaging Behav ; 11(3): 640-648, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26961091


Methylene blue USP (MB) is a FDA-grandfathered drug used in clinics to treat methemoglobinemia, carbon monoxide poisoning and cyanide poisoning that has been shown to increase fMRI evoked blood oxygenation level dependent (BOLD) response in rodents. Low dose MB also has memory enhancing effect in rodents and humans. However, the neural correlates of the effects of MB in the human brain are unknown. We tested the hypothesis that a single low oral dose of MB modulates the functional connectivity of neural networks in healthy adults. Task-based and task-free fMRI were performed before and one hour after MB or placebo administration utilizing a randomized, double-blinded, placebo-controlled design. MB administration was associated with a reduction in cerebral blood flow in a task-related network during a visuomotor task, and with stronger resting-state functional connectivity in multiple regions linking perception and memory functions. These findings demonstrate for the first time that low-dose MB can modulate task-related and resting-state neural networks in the human brain. These neuroimaging findings support further investigations in healthy and disease populations.

Encéfalo/efeitos dos fármacos , Azul de Metileno/farmacologia , Psicotrópicos/farmacologia , Administração Oral , Adulto , Encéfalo/diagnóstico por imagem , Encéfalo/fisiologia , Mapeamento Encefálico , Circulação Cerebrovascular/efeitos dos fármacos , Método Duplo-Cego , Feminino , Humanos , Imagem por Ressonância Magnética , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Vias Neurais/diagnóstico por imagem , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Testes Neuropsicológicos , Descanso , Percepção Visual/efeitos dos fármacos , Percepção Visual/fisiologia