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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 multipotency/stemness marker Nestin expression. hNPCs were mock infected or infected with SINV at m.o.i 1 (A,B) At 48 hour after infection, expression of stemness marker Nestin was analyzed by flow cytometry. A representative blot is shown in (A). Statistics from three repeats were shown in (B), samples were gated on Nestin staining positive cells, and percentage of Nestin-positive cells in SINV infected sample were shown. Error bars indicate SDs. *, statistical significance (p < 0.05) compared to the mock infected control. (C,D) Western Blot analysis of the expression of Nestin during the infection time course. Each experiment was performed at least three times. A representative blot is shown in (C). Blots were scanned, and relative expression levels of proteins were normalized to GAPDH and shown in (D). Error bars indicated SDs. *, statistical significance (p < 0.05) compared to the mock.
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Fig3: Effect of SINV infection on hNPCs multipotency/stemness marker Nestin expression. hNPCs were mock infected or infected with SINV at m.o.i 1 (A,B) At 48 hour after infection, expression of stemness marker Nestin was analyzed by flow cytometry. A representative blot is shown in (A). Statistics from three repeats were shown in (B), samples were gated on Nestin staining positive cells, and percentage of Nestin-positive cells in SINV infected sample were shown. Error bars indicate SDs. *, statistical significance (p < 0.05) compared to the mock infected control. (C,D) Western Blot analysis of the expression of Nestin during the infection time course. Each experiment was performed at least three times. A representative blot is shown in (C). Blots were scanned, and relative expression levels of proteins were normalized to GAPDH and shown in (D). Error bars indicated SDs. *, statistical significance (p < 0.05) compared to the mock.

Mentions: To investigate the induction of apoptosis upon SINV infection, at 24 h.p.i mock infected and SINV infected cells were fixed, stained for active-caspase 3, and analyzed by flow cytometry. Compared to uninfected cells, a 2-fold increase in the percentage of active-caspase 3 positive cells in SINV infected culture was observed (Mock 22.5% ± 1.25% vs. SINV infected 35% ± 0.33%) (Figure 3C). To further characterize apoptotic events in hNPCs culture upon SINV infection, the expression of active-caspase 3 was monitored on a protein level by Western Blot throughout the infection time course (Figure 2D). Expression of active-caspase 3 was significantly elevated at 24–48 h.p.i, consistent with our observations of CPE (Figure 2E). This result clearly showed that SINV induces apoptosis, and therefore cell death, in hNPCs.


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 multipotency/stemness marker Nestin expression. hNPCs were mock infected or infected with SINV at m.o.i 1 (A,B) At 48 hour after infection, expression of stemness marker Nestin was analyzed by flow cytometry. A representative blot is shown in (A). Statistics from three repeats were shown in (B), samples were gated on Nestin staining positive cells, and percentage of Nestin-positive cells in SINV infected sample were shown. Error bars indicate SDs. *, statistical significance (p < 0.05) compared to the mock infected control. (C,D) Western Blot analysis of the expression of Nestin during the infection time course. Each experiment was performed at least three times. A representative blot is shown in (C). Blots were scanned, and relative expression levels of proteins were normalized to GAPDH and shown in (D). Error bars indicated SDs. *, statistical significance (p < 0.05) compared to the mock.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4307679&req=5

Fig3: Effect of SINV infection on hNPCs multipotency/stemness marker Nestin expression. hNPCs were mock infected or infected with SINV at m.o.i 1 (A,B) At 48 hour after infection, expression of stemness marker Nestin was analyzed by flow cytometry. A representative blot is shown in (A). Statistics from three repeats were shown in (B), samples were gated on Nestin staining positive cells, and percentage of Nestin-positive cells in SINV infected sample were shown. Error bars indicate SDs. *, statistical significance (p < 0.05) compared to the mock infected control. (C,D) Western Blot analysis of the expression of Nestin during the infection time course. Each experiment was performed at least three times. A representative blot is shown in (C). Blots were scanned, and relative expression levels of proteins were normalized to GAPDH and shown in (D). Error bars indicated SDs. *, statistical significance (p < 0.05) compared to the mock.
Mentions: To investigate the induction of apoptosis upon SINV infection, at 24 h.p.i mock infected and SINV infected cells were fixed, stained for active-caspase 3, and analyzed by flow cytometry. Compared to uninfected cells, a 2-fold increase in the percentage of active-caspase 3 positive cells in SINV infected culture was observed (Mock 22.5% ± 1.25% vs. SINV infected 35% ± 0.33%) (Figure 3C). To further characterize apoptotic events in hNPCs culture upon SINV infection, the expression of active-caspase 3 was monitored on a protein level by Western Blot throughout the infection time course (Figure 2D). Expression of active-caspase 3 was significantly elevated at 24–48 h.p.i, consistent with our observations of CPE (Figure 2E). This result clearly showed that SINV induces apoptosis, and therefore cell death, in hNPCs.

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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Related in: MedlinePlus