Cellular responses to Sindbis virus infection of neural progenitors derived from human embryonic stem cells

Cellular responses to Sindbis virus infection of neural progenitors derived from human embryonic stem cells Xu et al. Xu et al. BMC Research Notes 2014, 7:757 http://www.biomedcentral.com/1756-0500/7/757 Xu et al. BMC Research Notes 2014, 7:757 http://www.biomedcentral.com/1756-0500/7/757 SHORT REPORT Open Access Cellular responses to Sindbis virus infection of neural progenitors derived from human embryonic stem cells Jie Xu1, Rodney J Nash1 and Teryl K Frey1,2* Abstract Background: Sindbis virus (SINV) causes age-dependent encephalitis in mice, and therefore serves as a model to study viral encephalitis. SINV is used as a vector for the delivery of genes into selected neural stem cell lines; however, the toxicity and side effects of this vector have rarely been discussed. In this context, we investigated the cellular responses of human embryonic stem cell (hESCs) derived neural progenitors (hNPCs) to SINV infection by assessing susceptibility of the cells to SINV infection, analyzing the effect of infection on cell proliferation and cell death, and examining the impact of SINV infection on hNPCs markers of stemness. Findings: We found that hNPCs are highly susceptible to SINV infection. Upon infection, the viruses induced apoptosis to hNPCs while not affecting the expression of cell proliferation markers. Lastly, SINV infections result in significant changes in the expression of key regulators of hNPCs’ plasticity and homeostasis. Conclusion: The robust and versatile signaling, proliferation, and other cell responses of hESCs-derived hNPCs to virus infection demonstrated that it is a good model to study the pathogenesis of viral-induced neurodevelopmental and degenerative diseases. On the other hand, the toxicity of SINV to hNPCs cells cannot be ignored, and therefore extra care should be taken when using SINV as a vector to deliver genes into human stem cell lines. Keywords: Stem cell infection, Human neural progenitors, Sindbis virus Background Sindbis virus (SINV), a positive strand RNA virus in the genus Alphavirus of the Togaviridae family, causes rash and fever in humans and age-dependent encephalitis in mice. The virus has long served as a model to study viral encephalitis induced by viruses in the Togaviridae family, as well as other neurovirulent viruses [1,2]. More importantly, SINV has been widely used to express a variety of genes in cultured neurons and in vivo as it provides fast onset and high level of expression of foreign genes [3]. The high and rapid expression of foreign genes from SINV vector is accomplished at the cost of shutting off protein nsP2 synthesis in the infected host cells. As nsP2 are commonly encoded in the SINV vector, whether this machinery leads to the toxicity of the SINV vector in cells are unknown [4]. In recent studies, * Correspondence: 1 Department of Biology, Georgia State University, Atlanta, GA, USA 2 Jeevan Bioscience, Inc., Dunwoody, GA, USA SINV was used as vector to deliver HIV gp120 into hNPCs and showed a lytic effect to cells, while others using wild-type HIV did not [5,6]. The mechanisms governing pathogenic outcome and extent of SINV replication in human cells are not well characterized [7-9]. As SINV gains popularity in neurotherapy as an ideal vector for gene transfer into neural stem/progenitor cells, the toxicity, as well as other side effects of these vectors, needs to be addressed [3,10]. Recently, hNPCs have been developed commercially as a model to study developmental neurotoxicity and neurotherapy [11]. In this study, we used hNP1 cells (ArunA Biomedical), an hNPC line derived from the NIHapproved H9 (WiCell Research Institute’s H9 (WA09)) human embryonic stem cell line [12]. Undifferentiated hNP1 cells stably express the stemness markers Nestin and SOX2, and the cells have the capacity to differentiate into multiple neuronal subtypes, including cholinergic, dopaminergic, and GABAergic neurons, glia, and oligodendrocytes. hNP1 cells have not been immortalized or © 2014 Xu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. BMC Research Notes 2014, 7:757 http://www.biomedcentral.com/1756-0500/7/757 otherwise transformed, and therefore the potential caveats of transformation are not an issue when using these cells. In addition, hNP1 can grow as a monolayer without fibroblast support, another advantage over other primary neuronal stem cells. Lastly, hNP1 cells have been successfully used in the study of radiation sensitivity, neurotoxicity screening, neurophysiology, tissue engineering, and translational medicine [13-19]. Better still, hNP1 cells have been recently applied to neurotherapies of multiple sclerosis patients; techniques such as SINV-based vectormediated gene transfer were intensively utilized [20]. Thus, the study of the cellular responses of hNP1 cells to SINV infection will serve as a reference for the application of commercialized hNPCs in stem cell infection and translational medicine field. Here, we show that hNPCs are highly susceptible to SINV infection with a cytopathic effect (CPE) starting at 24 hours post infection (h.p.i). SINV replicated and disseminated effectively as virus titer increased by 100 fold in hNPC cells, and the percentage of infected cells reached over 85% at later time points. Besides these developments, the virus also triggered apoptosis while not affecting the expression of cell proliferation markers. In addition, reduced expression of hNPCs stemness marker Nestin was observed throughout the infection time course. Close scrutiny suggested that SINV upsets the dedicated balance of hNPC cell signaling, such as STAT3, but not NF-kB and pIRF3. Thus, we added this commercialized hNPC line into another type of human neural stem cells that are susceptible to SINV infection. SINV establish lytic replication cycles in hNPCs, and thus extra care should be taken when using SINV-based vector for gene delivery in stem cell therapy. Page 3 of 10 Modified Eagle Medium (D-MEM, Cellgro) with 5% fetal bovine serum (FBS), as described previously [21]. Virus stock preparation To prepare SINV stock, 80% confluent BHK cells were infected with the SINV HR strain at a multiplicity of infection (m.o.i) of 0.1. Culture medium was collected at 2 days post infection (d.p.i) when CPE was obvious in the culture. Cell-free (clarified) virus stock was prepared by collecting supernatant of such medium after high speed centrifugation. hNP1 cells were infected as follows: after the removal of culture media, cells were washed once with pho (...truncated)