Human Embryonic Stem Cells-Derived Cardiomyocytes are susceptible to Group B Coxsackie Viruses infection and respond to Type I INF-α treatment

SCASSA, MARÍA E.; QUESTA, MARÍA; CIFUENTE, JAVIER; JAQUENOD DE GIUSTI, CAROLINA; BLUGUERMANN, CAROLINA; ROMORINI, LEONARDO; VIDELA RICHARDSON, GUILLERMO; FERNANDEZ ESPINOSA, DAMIAN D.; SEVLEVER, GUSTAVO E.; GOMEZ, RICARDO M.; MIRIUKA, SANTIAGO

California, EEUU

Congreso; 8th Annual Meeting International Society for Stem Cell Research; 2010

International Society for Stem Cell Research

Background: Human embryonic stem cells (hESCs) are pluripotent cells with the ability to self-renew and differentiate to a wide range of specialized cell types, including cardiomyocytes. Group B Coxsackie viruses (CSVB) are well-known for producing acute myocarditis and chronic dilated cardiomyopathy, but to the present day an effective specific therapy is still not available. The above considerations and the lack of a suitable human in vitro model for the study of myocarditis prompted us to determine if cardiomyocytes derived from hESCs may well represent a suitable model to study coxsackieviruses infections.Methods: Human cells lines WA-09, HUES-5 and HUES-16 were used. Using an embryoid body-based approach, we detected the development of cardiac lineage in the differentiation cultures, by the appearance of spontaneously contracting areas. mRNA and protein analysis of these areas revealed the expression of the following cardiac markers: mesoderm posterior 1, Nkx2.5, Islet-1, GATA-4, cardiac troponin T, atrial natriuretic peptide and α-myosin heavy chain. Moreover, these cells display functional similarities to their adult counterparts. For viral infection, we used two variants of CSVB1 (M and N). hESCs and Embryoid Bodies (EBs) were subjected to viral infection at day 0 (undifferentiated state) and at day 14 after the onset of differentiation.Results: First, we showed the presence of CAR (coxsackie-adenovirus receptor) and DAF (decay accelerating factor) in all cell lines, both by RT-PCR and immunocytochemistry. These membrane proteins are required for a productive CSVB infectious cycle, acting as an internalization receptor, and a co-receptor, respectively. We also showed the presence of TLR-3 (toll-like receptor-3) and type I Interferon (IFN I) receptors I and II, both involved in the immune response against the virus, by RT-PCR. In all cases, gene and protein expression were determined at day 0 and day 14. These findings led us to investigate the susceptibility to infection of hESCs and EBs by two variants of CSVB1 (M and N). This was assessed by determining the presence of cytopathic effects. Cellular ballooning was visible as soon as 12 hours post-infection and was followed by cessation of beating activity, which preceded cell death, and typically was observed at 36-48 hours post-infection. Furthermore, culture supernatant infectivity reached 104-105 plaque-forming units per ml and capsid protein VP1 was abundant in infected cells. Finally, given the relevance of IFN I in innate immune response against viral infection, we treated cells with 100 U/ml of recombinant human IFN I-β prior to viral infection. We observed that this potent antiviral cytokine conferred protection to undifferentiated hESCs, as judged by the significantly reduced cytopathic effect observed.Conclusions: In this study we found that hESC the tested cell lines are susceptible to CSVB infection, both at the undifferentiated (pluripotent hESCs) and the differentiated stages (hESC-derived EBs) and respond to IFN I-β treatment by reducing viral activity. As a whole, we propose that hESCs and contractile hESC-derived EBs would constitute suitable in vitro models to study molecular mechanisms involved in CSVB-cardiac cell interaction, and to test potential antiviral drugs in order to gain insights into the development of effective therapies.