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

Xu J, Nash RJ, Frey TK - BMC Res Notes (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Georgia State University, Atlanta, GA, USA. tfrey@gsu.edu.

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.

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hNPCs are highly susceptible to SINV infection. hNPCs were infected with SINV at an m.o.i. of 1 (A) At 4, 8, 12, 24, and 48 hours after infection, cells were immunostained with antibodies against tubulin (green) and the SINV nonstructural proteins (SINV_NSP, red). Nuclei were counterstained with DAPI (blue). Bars: 10 um. (B) Based on SINV_NSP immune-staining as shown in Figure 1A, the percentage of infected hNPCs was determined at each time point. The results are the means of two independent experiments; at least 200 cells from three different fields were counted at each time point. (C) hNPC (hNP1) and BHK cells were infected at an m.o.i of 1. Medium collected at the same time points was titered for infectious virus. Each data point is the average of duplicate titration from three experiments. Error bars indicate SDs. *, statistical significance (p < 0.05) in comparison with the SINV-infected sample of the same time point.
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Fig1: hNPCs are highly susceptible to SINV infection. hNPCs were infected with SINV at an m.o.i. of 1 (A) At 4, 8, 12, 24, and 48 hours after infection, cells were immunostained with antibodies against tubulin (green) and the SINV nonstructural proteins (SINV_NSP, red). Nuclei were counterstained with DAPI (blue). Bars: 10 um. (B) Based on SINV_NSP immune-staining as shown in Figure 1A, the percentage of infected hNPCs was determined at each time point. The results are the means of two independent experiments; at least 200 cells from three different fields were counted at each time point. (C) hNPC (hNP1) and BHK cells were infected at an m.o.i of 1. Medium collected at the same time points was titered for infectious virus. Each data point is the average of duplicate titration from three experiments. Error bars indicate SDs. *, statistical significance (p < 0.05) in comparison with the SINV-infected sample of the same time point.

Mentions: To determine the permissiveness of hNPCs to SINV infection, we plated them onto Matrigel-coated plates and infected them with SINV at an m.o.i of 1. The capacity of the virus to infect, replicate, and disseminate in hNPCs was evaluated by immunofluorescence 4, 8, 12, 24, and 48 h following infection using an antibody directed towards the viral nonstructural protein SINV-NSP. In addition, we used an antibody towards tubulin to monitor cell morphology changes during the infection (Figure 1A). At 4 h following infection, a number of cells, albeit a small number, stained positive for SINV-NSP, revealing their permissiveness to SINV infection. No significant changes in cell shape/morphology were detected at this point. The observation of cultures from 4 to 24 h after infection showed that while only 3.8% ±0.32% of the cells were infected at 4 h.p.i, a large proportion of cells (86.8% ±0.8%) did so by 24 h.p.i (Figure 1B), demonstrating that the virus replicates in hNPCs and disseminates efficiently. CPE (i.e. presence of floaters, elongation of adherent cell bodies) was detected at 24 h.p.i, and at 48 h, few cells remained attached to the plate compared to uninfected controls. The percentage of infected cells no longer increased at 48 h.p.i, probably due to the massive loss of proliferating hNPCs upon SINV infection. Interestingly, elongated cell morphology was noticed in infected cells at this time point, indicating that SINV may induce premature differentiation of hNPCs or otherwise remove the capacity to maintain stemness. Extracellular virus yield was examined as a measurement of SINV replication efficiency (Figure 1C). Virus titers increased by 100 fold from 4 h.p.i (103pfu/ml) to 24 h.p.i (105pfu/ml), at which time the virus had disseminated throughout most of the culture. The viral titer achieved by SINV in hNPCs is 2–3 log lower than those seen in BHK cells (107 or 108 pfu/cell).Figure 1


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

Xu J, Nash RJ, Frey TK - BMC Res Notes (2014)

hNPCs are highly susceptible to SINV infection. hNPCs were infected with SINV at an m.o.i. of 1 (A) At 4, 8, 12, 24, and 48 hours after infection, cells were immunostained with antibodies against tubulin (green) and the SINV nonstructural proteins (SINV_NSP, red). Nuclei were counterstained with DAPI (blue). Bars: 10 um. (B) Based on SINV_NSP immune-staining as shown in Figure 1A, the percentage of infected hNPCs was determined at each time point. The results are the means of two independent experiments; at least 200 cells from three different fields were counted at each time point. (C) hNPC (hNP1) and BHK cells were infected at an m.o.i of 1. Medium collected at the same time points was titered for infectious virus. Each data point is the average of duplicate titration from three experiments. Error bars indicate SDs. *, statistical significance (p < 0.05) in comparison with the SINV-infected sample of the same time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307679&req=5

Fig1: hNPCs are highly susceptible to SINV infection. hNPCs were infected with SINV at an m.o.i. of 1 (A) At 4, 8, 12, 24, and 48 hours after infection, cells were immunostained with antibodies against tubulin (green) and the SINV nonstructural proteins (SINV_NSP, red). Nuclei were counterstained with DAPI (blue). Bars: 10 um. (B) Based on SINV_NSP immune-staining as shown in Figure 1A, the percentage of infected hNPCs was determined at each time point. The results are the means of two independent experiments; at least 200 cells from three different fields were counted at each time point. (C) hNPC (hNP1) and BHK cells were infected at an m.o.i of 1. Medium collected at the same time points was titered for infectious virus. Each data point is the average of duplicate titration from three experiments. Error bars indicate SDs. *, statistical significance (p < 0.05) in comparison with the SINV-infected sample of the same time point.
Mentions: To determine the permissiveness of hNPCs to SINV infection, we plated them onto Matrigel-coated plates and infected them with SINV at an m.o.i of 1. The capacity of the virus to infect, replicate, and disseminate in hNPCs was evaluated by immunofluorescence 4, 8, 12, 24, and 48 h following infection using an antibody directed towards the viral nonstructural protein SINV-NSP. In addition, we used an antibody towards tubulin to monitor cell morphology changes during the infection (Figure 1A). At 4 h following infection, a number of cells, albeit a small number, stained positive for SINV-NSP, revealing their permissiveness to SINV infection. No significant changes in cell shape/morphology were detected at this point. The observation of cultures from 4 to 24 h after infection showed that while only 3.8% ±0.32% of the cells were infected at 4 h.p.i, a large proportion of cells (86.8% ±0.8%) did so by 24 h.p.i (Figure 1B), demonstrating that the virus replicates in hNPCs and disseminates efficiently. CPE (i.e. presence of floaters, elongation of adherent cell bodies) was detected at 24 h.p.i, and at 48 h, few cells remained attached to the plate compared to uninfected controls. The percentage of infected cells no longer increased at 48 h.p.i, probably due to the massive loss of proliferating hNPCs upon SINV infection. Interestingly, elongated cell morphology was noticed in infected cells at this time point, indicating that SINV may induce premature differentiation of hNPCs or otherwise remove the capacity to maintain stemness. Extracellular virus yield was examined as a measurement of SINV replication efficiency (Figure 1C). Virus titers increased by 100 fold from 4 h.p.i (103pfu/ml) to 24 h.p.i (105pfu/ml), at which time the virus had disseminated throughout most of the culture. The viral titer achieved by SINV in hNPCs is 2–3 log lower than those seen in BHK cells (107 or 108 pfu/cell).Figure 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Georgia State University, Atlanta, GA, USA. tfrey@gsu.edu.

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.

Show MeSH
Related in: MedlinePlus