Limits...
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.

Show MeSH

Related in: MedlinePlus

Western Blot analyzes of the expression of key regulator of differentiation, inflammation responses. The expression of cellular proteins that regulate key events during differentiation, as well as early inflammation responses, were analyzed during infection time course. Each experiment was performed at least twice. A representative blot was shown. GAPDH: loading control. (B) Quantification of western blotting on proteins tested in (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Western Blot analyzes of the expression of key regulator of differentiation, inflammation responses. The expression of cellular proteins that regulate key events during differentiation, as well as early inflammation responses, were analyzed during infection time course. Each experiment was performed at least twice. A representative blot was shown. GAPDH: loading control. (B) Quantification of western blotting on proteins tested in (A).

Mentions: Finally, we wanted to investigate the mechanism behind SINV modulation of hNPCs morphology, proliferation and multipotency/stemness. Expression of multiple signaling molecules (NF-kB p65, pSTAT3, pIRF3 and pERK1/2) on crucial regulation pathways during SINV infection course were analyzed by Western Blot (Figure 4A and B). The expression of active phosphorylated (Y705) STAT3 (pSTAT3) was down-regulated by SINV at 24 h.p.i, suggesting a negative regulation of JAK/STAT pathway upon infection. pSTAT3 is required for normal hNPC differentiation, and the reduction in this protein possibly led to impaired differentiation potential [25]. Expression of Phospho-p44/42 MAPK (pERK1/2) was similarly down-regulated by SINV at 24 h.p.i. The MAPK pathway regulates multiple phosphorylation events, including those involved in cell proliferation. Robust expression of pERK1/2 was shown to be pro-survival [26]. Therefore, it is highly possible that SINV impairs hNPC proliferation by repressing pERK1/2 expression, and therefore the regulation of the MAPK pathway. Expression of NF-kB p65 and phospho-interferon regulation factor 3 (pIRF3) were not significantly altered upon SINV infection. As both proteins serve as signaling molecules in IFN induction, it was plausible to suggest SINV did not induce high expression of cytokines, and therefore the inflammatory response. Expression of all four proteins was significantly decreased at 48 h.p.i, possibly due to massive loss of cells at this time point (Figure 4B).Figure 4


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)

Western Blot analyzes of the expression of key regulator of differentiation, inflammation responses. The expression of cellular proteins that regulate key events during differentiation, as well as early inflammation responses, were analyzed during infection time course. Each experiment was performed at least twice. A representative blot was shown. GAPDH: loading control. (B) Quantification of western blotting on proteins tested in (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Western Blot analyzes of the expression of key regulator of differentiation, inflammation responses. The expression of cellular proteins that regulate key events during differentiation, as well as early inflammation responses, were analyzed during infection time course. Each experiment was performed at least twice. A representative blot was shown. GAPDH: loading control. (B) Quantification of western blotting on proteins tested in (A).
Mentions: Finally, we wanted to investigate the mechanism behind SINV modulation of hNPCs morphology, proliferation and multipotency/stemness. Expression of multiple signaling molecules (NF-kB p65, pSTAT3, pIRF3 and pERK1/2) on crucial regulation pathways during SINV infection course were analyzed by Western Blot (Figure 4A and B). The expression of active phosphorylated (Y705) STAT3 (pSTAT3) was down-regulated by SINV at 24 h.p.i, suggesting a negative regulation of JAK/STAT pathway upon infection. pSTAT3 is required for normal hNPC differentiation, and the reduction in this protein possibly led to impaired differentiation potential [25]. Expression of Phospho-p44/42 MAPK (pERK1/2) was similarly down-regulated by SINV at 24 h.p.i. The MAPK pathway regulates multiple phosphorylation events, including those involved in cell proliferation. Robust expression of pERK1/2 was shown to be pro-survival [26]. Therefore, it is highly possible that SINV impairs hNPC proliferation by repressing pERK1/2 expression, and therefore the regulation of the MAPK pathway. Expression of NF-kB p65 and phospho-interferon regulation factor 3 (pIRF3) were not significantly altered upon SINV infection. As both proteins serve as signaling molecules in IFN induction, it was plausible to suggest SINV did not induce high expression of cytokines, and therefore the inflammatory response. Expression of all four proteins was significantly decreased at 48 h.p.i, possibly due to massive loss of cells at this time point (Figure 4B).Figure 4

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