Wieloelektrodowa sieć pomiarowa i badania immunofluorescencyjne do oceny wpływu zakażenia HHV-6 na różnicowane komórki macierzyste indukowane
A multi-electrode array and immunofluorescence workflow to characterize impacts of HHV-6 infection in differentiated induced pluripotent stem cells
W skrócie
[Preprint - wstępne wyniki] Naukowcy opracowali nową metodę badania, jak wirus HHV-6 (herpeswirus ludzkiej rodziny Roseolovirus) wpływa na pracę komórek nerwowych. Using sztucznych komórek nerwowych wzrastanych na specjalnych płytkach z wbudowanymi elektrodami, wykazali, że zakażenie HHV-6A zmienia elektryczną aktywność neuronów w porównaniu do komórek niezakażonych. Ta metoda może pomóc naukowcom w przyszłości badać, w jaki sposób wirusy zmieniają pracę mózgu i mieć może znaczenie dla zrozumienia epilepsji i innych chorób neurologicznych związanych z HHV-6.
Oryginalny abstract (angielski)
ABSTRACT Roseoloviruses, notably human herpesviruses 6A and 6B (HHV-6A and HHV-6B), are neurotropic viruses implicated as agents in neurological disorders, including: epilepsy, multiple sclerosis, and chronic fatigue syndrome. However, the effects of roseolovirus infection on neuronal signaling and network activity are not characterized. This is, in part, due to the complexities of monitoring electrical activity in individual neurons and across neuronal connections during viral infection. This protocol describes a human induced pluripotent stem cell (iPSC)-derived neuronal culture system that employs multi-electrode array (MEA) recordings to study neurophysiological changes during viral infection. Two culture platforms are described: (a) NGN2-induced forebrain neuronal cultures; and, (b) progenitor cell-derived neuron–astrocyte mixed cultures. Cell composition in cultures is validated by immunofluorescence staining using neuronal, glial, and viral markers. Functional activity is assessed using extracellular recordings detected via the MEA2100 system. Mean firing rate, inter-spike interval, single-electrode bursting, and network burst activity are characterized between roseolovirus-infected versus uninfected (control) states. Pharmacological treatment with bicuculline, gabazine, and nicotine in conjunction with immunofluorescence is used to confirm functional responsiveness of defined neuronal neurotransmitter chemotypes. Here, we show that HHV-6A infection changes neuronal firing compared to uninfected controls. The methods/workflows described provide a strategy to study how virus infection alters neuronal excitability and may be adapted to compare the effects of different viruses on nerve cell function, infection time courses, different multiplicities of infection (MOI), and therapeutic interventions. SUMMARY Culturing nerve cells on embedded electrode array plates combined with parallel cultures on standard plates (and flasks) for immunofluorescence is an effective method for characterizing the impacts of virus infection on neuronal electrogenic function. Here we introduce a workflow to characterize the electrophysiological impacts of herpesvirus infection on cultured neurons.