Susceptibility Of Immortalized Human Umbilical Cord Mesenchymal Stem Cells To Common Viruses Infection

Umbilical cord derived Mesenchymal stem cells have stronger ability of amplification, lower immunogenicity compared to bone marrow derived MSCs and possess multipotential differentiation, high safety. Therefore, MSCs hold extensive promise for clinical application in regenerative medicine, gene therapy, cell therapy,vaccine production and verification, etc. Our group had constructed immortalized human umbilical cord mesenchymal stem cells by introducing human telomerase catalytic subunit into human umbilical cord mesenchymal stem cells in previous studies, and the cell line displays the potential of directional differentiation in vitro and maintained normal diploid karyotype. In addition, the surface markers of the cell line accords with MSCs and displays no oncogenicity in mice. Based on the immortalized hMSCs in previous studies, this research subject aims to detect susceptibility of hMSCs to the following 10 kinds of virus among six families:Vaccinia tian tan(VTT), Human cytomegalovirus(HCMV), Enterovirus type71(EV71), Recombinant adenovirus(rAd5-GFP, r AdF35-GFP), Recombinant Sendai virus(rSeV-HIVgag),A/California/7/2009(H1N1),A/Brisbane/10/2007(H3N2),A/Brisb ane/59/2007(H1N1),(B/Florida/4/2006, B/Yamagata line), to lay the foundation for the further application of the cell line.hMSCs were infected with different doses of the virus regarding the susceptible cells of the virus as positive control at the same time, then viral infection was determined by observing virus-induced cytopathic effect(CPE) or the report gene expression of green fluorescent protein through the microscope and detecting viral antigen with indirect immunofluorescence technique; based on the results above, the minimum amount of the virus which could infect hMSCs was determined; in addition,the amount of virus released by infected cells was measured and compared by plaque assay with crystal violet staining, real-time polymerase chain reaction(PCR) detection,etc. The results showed that VTT and HCMV caused apparent cytopathic effect after infecting hMSCs; green fluorescent could be observed on the hMSCs infected with r Ad5-GFP and rAd5F35-GFP; indirect immunofluorescence assay showed that there was obvious green fluorescent on the hMSCs infected with VTT, HCMV,r SeV-HIVgag,(B/Florida/4/2006, B/Yamagata line) and A/Brisbane/10/2007(H3N2),but not on the hMSCs infected with EV71, A/California/7/2009(H1N1),A/Brisbane/59/2007(H1N1), which indicates that hMSCs are permissive to infectionwith the former 7 kinds of virus, but not with the latter 3 kinds of virus. The minimum multiplicity of infection(MOI) results showed that the minimum MOIs that VTT,HCMV, rSeV-HIVgag, rAd5-GFP, rAd5F35-GFP, A/Brisbane/10/2007(H3N2),(B/Florida/4/2006, B/Yamagata line) infected hMSCs were 1.5×10-5, 0.01, 5×10-5,150, 2, 1, 5×10-4 respectively, and relatively positive controls were 1.5×10-5, 2×10-4,5×10-5, 5×10-5, 2×10-5, 0.01, 5×10-4, stating that hMSCs have very different susceptibility to various viruses, but the lowest dose of infection is generally higher than control cells. On that basis, detected progeny virus released by infected cells and found that after infected with equivalent VTT for 48 h and 72 h, the viral titers in supernatant of hMSCs were 7.13×103, 2.30×105 PFU/mL respectively, while the viral titers in supernatant of control cells were 3.5×105, 2.7×106 PFU/mL; After 4-8 d of infection with equivalent HCMV, the viral DNA copies in supernatant of hMSCs were 3.919、4.240、4.408、4.774、4.730 lgCopies/mL respectively, and the viral DNA copies in supernatant of control cells were 4.062、 4.069、 4.548、 6.017、 6.904lgCopies/m L;After 1-3 d of infection with equivalent(B/Florida/4/2006, B/Yamagata line), the viral HAs in supernatant of hMSCs were 0、1:2、1:2 respectively, and the HAs in supernatant of control cells were all 1:256.In conclusion, this study preliminary explored susceptibility of hMSCs to common 10 kinds of strains among six families, then analyzed and compared the minimum amount of the virus which could infect hMSCs and the amount of virus release, in order to provide an experimental foundation for the application of hMSCs in vaccine production and identification, gene therapy, cell therapy, etc.