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Activation of apoptotic signalling events in human embryonic stem cells upon Coxsackievirus B3 infection

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Abstract

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate to a wide range of specialized cells and hold great promise as models for human development and disease, as well as for drug discovery and cell-replacement therapies. Group B Coxsackie viruses (CVBs) produce acute myocarditis, pancreatitis, non-septic meningitis and encephalitis in neonates, children and young adults. Moreover, CVBs can produce spontaneous miscarriage after early embryo infection. It was reported that hESCs express CVBs receptors and are susceptible to CVB3 infection. Apoptosis is one of the hallmarks of CVBs infection although details regarding CVB3 involvement in the apoptotic processes remain elusive. In order to evaluate the mechanisms of cell death induced by CVB3 in these pluripotent cells, we infected HUES-5 (H5) and WA01 (H1) hESC lines with CVB3. After validating the maintenance of stemness in these hESC lines when grown as confluent monolayers in feeder-free conditions, we analysed several aspects of programmed cell death triggered by CVB3. In all cases, we detected chromatin condensation, DNA fragmentation and caspase-9 and 3 cleavages. Moreover, we observed the presence of cleaved PARP product which was preceded by the appearance of p17, the catalytically active fragment of caspase-3. Mitochondrial function assays revealed a MOI dependent decrease in cell viability at 24 h post-infection (pi). No appreciable modifications in Bcl-2, Bcl-XL and Bax protein levels were observed upon CVB3 infection during 5–24 h observation period. However, a marked decrease in pro-apoptotic Bad abundance was detected without changes in its mRNA levels. In this study we found that the hESCs are highly susceptible to CVB3 infection and display elevated apoptosis rates, thus emerging as suitable human non-transformed in vitro models to study CVB3-induced apoptosis and resulting relevant to understand CVBs pathogenesis.

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Abbreviations

hESCs:

Human embryonic stem cells

CVB3:

Coxsackie virus B3

H5:

HUES-5 cell line

H1:

WA01 cell line

MOI:

Multiplicity of infection

PARP:

Poly-ADP-ribose-polymerase

CAR:

Coxsackievirus-adenovirus receptor

DAF:

Decay accelerating factor

VP1:

Viral protein 1

Ac-DEVDpNA :

Acetyl-Asp-Glu-Val-Asp-7-amino-4 p-nitroanilide

iMEF:

Inactivated mouse embryonic fibroblast

KSR:

Knockout Serum Replacement

DAPI:

4-6-Diamidino-2-phenylindole

XTT:

2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5 [(phenylamino) carbonyl]-2 H-tetrazolium hydroxide

PMS:

N-methyl dibenzopyrazine methyl sulfate

BCA:

Bicinchoninic acid

Pi:

Post-infection

Cpe:

Cytopathic effect

TUNEL:

Terminal dUTP nick end labeling

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Acknowledgments

This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PID2007-00112, PICT 07-00642 and PICT 07-00028. The authors would like to thank Marcela Cañari for her skillful assistance.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Laboratorio de Biología del Desarrollo Celular, LIAN, FLENI, Ruta 9, Km 52,5, Escobar, B1625XAF, Buenos Aires, Argentina

    Leonardo Romorini, María E. Scassa, Guillermo Videla Richardson, Carolina Blüguermann, María Questa, Damián D. Fernandez Espinosa, Gustavo E. Sevlever & Santiago G. Miriuka

  2. Instituto de Biotecnología y Biología Molecular, CCT-La Plata, CONICET-UNLP, Calle 49 y 115, 1900 La Plata, Buenos Aires, Argentina

    Carolina Jaquenod de Giusti & Ricardo M. Gómez

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  1. Leonardo Romorini
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Correspondence to Santiago G. Miriuka.

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Romorini, L., Scassa, M.E., Richardson, G.V. et al. Activation of apoptotic signalling events in human embryonic stem cells upon Coxsackievirus B3 infection. Apoptosis 17, 132–142 (2012). https://doi.org/10.1007/s10495-011-0668-z

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