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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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Effect of SINV infection on hNPCs proliferation and undifferentiated phenotype. hNPCs were mock infected or infected with SINV at m.o.i 1 (A, B) Cell proliferation was analyzed by an EdU incorporation assay. A representative blot is shown in (A), samples were gated on EdU staining positive cells, and percentage of EdU incorporated cells in SINV infected sample were shown. (B) Percentage of EdU incorporated cells in mock and SINV infected cells were quantified by flow cytometry. Error bars indicate SDs. *, statistical significance (p < 0.05) in comparison with mock. (C) At 48 hour after infection, expression of apoptotic cell marker active caspase 3 was analyzed by flow cytometry. This experiment was repeated at least twice, two titrations per experiment. Error bars indicate SDs. *, statistical significance (p < 0.05) compared to the mock infected control at same time point. (D, E) Western Blot analysis of the expression of active caspase 3 and proliferating cell marker PCNA during the infection time course. Each experiment was performed at least three times. A representative blot is shown in (D). Blots were scanned, and relative expression levels of proteins were normalized to GAPDH and shown in (E). Error bars indicated SDs. *, statistical significance (p < 0.05) compared to the mock.
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Fig2: Effect of SINV infection on hNPCs proliferation and undifferentiated phenotype. hNPCs were mock infected or infected with SINV at m.o.i 1 (A, B) Cell proliferation was analyzed by an EdU incorporation assay. A representative blot is shown in (A), samples were gated on EdU staining positive cells, and percentage of EdU incorporated cells in SINV infected sample were shown. (B) Percentage of EdU incorporated cells in mock and SINV infected cells were quantified by flow cytometry. Error bars indicate SDs. *, statistical significance (p < 0.05) in comparison with mock. (C) At 48 hour after infection, expression of apoptotic cell marker active caspase 3 was analyzed by flow cytometry. This experiment was repeated at least twice, two titrations per experiment. Error bars indicate SDs. *, statistical significance (p < 0.05) compared to the mock infected control at same time point. (D, E) Western Blot analysis of the expression of active caspase 3 and proliferating cell marker PCNA during the infection time course. Each experiment was performed at least three times. A representative blot is shown in (D). Blots were scanned, and relative expression levels of proteins were normalized to GAPDH and shown in (E). Error bars indicated SDs. *, statistical significance (p < 0.05) compared to the mock.

Mentions: Since SINV led to massive cell loss, we investigated whether the diminished hNPC population was due to SINV-induced cell death or cell cycle arrest. The effect of SINV on hNPC proliferation was quantitatively analyzed by EdU incorporation, which demonstrated a significant reduced cell growth in infected cultures (Figure 2A). At 12 h.p.i, no significant change in EdU labeling was noticed, however, at 24 h.p.i, this reduced drastically from 74.7% ±3.2% in uninfected controls to 33.4% ±1.25% in SINV infected hNPCs (Figure 2B). This negative effect on cell proliferation was not demonstrated until 24 hours after infection, in correlation with virus replication kinetics.


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)

Effect of SINV infection on hNPCs proliferation and undifferentiated phenotype. hNPCs were mock infected or infected with SINV at m.o.i 1 (A, B) Cell proliferation was analyzed by an EdU incorporation assay. A representative blot is shown in (A), samples were gated on EdU staining positive cells, and percentage of EdU incorporated cells in SINV infected sample were shown. (B) Percentage of EdU incorporated cells in mock and SINV infected cells were quantified by flow cytometry. Error bars indicate SDs. *, statistical significance (p < 0.05) in comparison with mock. (C) At 48 hour after infection, expression of apoptotic cell marker active caspase 3 was analyzed by flow cytometry. This experiment was repeated at least twice, two titrations per experiment. Error bars indicate SDs. *, statistical significance (p < 0.05) compared to the mock infected control at same time point. (D, E) Western Blot analysis of the expression of active caspase 3 and proliferating cell marker PCNA during the infection time course. Each experiment was performed at least three times. A representative blot is shown in (D). Blots were scanned, and relative expression levels of proteins were normalized to GAPDH and shown in (E). Error bars indicated SDs. *, statistical significance (p < 0.05) compared to the mock.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Effect of SINV infection on hNPCs proliferation and undifferentiated phenotype. hNPCs were mock infected or infected with SINV at m.o.i 1 (A, B) Cell proliferation was analyzed by an EdU incorporation assay. A representative blot is shown in (A), samples were gated on EdU staining positive cells, and percentage of EdU incorporated cells in SINV infected sample were shown. (B) Percentage of EdU incorporated cells in mock and SINV infected cells were quantified by flow cytometry. Error bars indicate SDs. *, statistical significance (p < 0.05) in comparison with mock. (C) At 48 hour after infection, expression of apoptotic cell marker active caspase 3 was analyzed by flow cytometry. This experiment was repeated at least twice, two titrations per experiment. Error bars indicate SDs. *, statistical significance (p < 0.05) compared to the mock infected control at same time point. (D, E) Western Blot analysis of the expression of active caspase 3 and proliferating cell marker PCNA during the infection time course. Each experiment was performed at least three times. A representative blot is shown in (D). Blots were scanned, and relative expression levels of proteins were normalized to GAPDH and shown in (E). Error bars indicated SDs. *, statistical significance (p < 0.05) compared to the mock.
Mentions: Since SINV led to massive cell loss, we investigated whether the diminished hNPC population was due to SINV-induced cell death or cell cycle arrest. The effect of SINV on hNPC proliferation was quantitatively analyzed by EdU incorporation, which demonstrated a significant reduced cell growth in infected cultures (Figure 2A). At 12 h.p.i, no significant change in EdU labeling was noticed, however, at 24 h.p.i, this reduced drastically from 74.7% ±3.2% in uninfected controls to 33.4% ±1.25% in SINV infected hNPCs (Figure 2B). This negative effect on cell proliferation was not demonstrated until 24 hours after infection, in correlation with virus replication kinetics.

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