Monitoring Stem Cells Transplantation By Hybrid Baculovirus-adeno-associated Viral Vector-mediated Radionuclide Reporter Gene Imaging System

Purpose:Noninvasively and quantitatively monitoring stem cells in vivo is important for optimizing stem cells transplantation therapy.In this study,we constructed and prepared novel hybrid baculovirus-adeno-associated virus?BV-AAV?,and explored the the feasibility of utilizing this hybrid viral vector-mediated radionuclide reporter gene imaging in stem cells transplantation monitoring.Furthermore,we modified the BV with Sleeping Beauty transposon gene?SB100×?,through which the potential of long-term reporter gene imaging was evaluated.Methods:Enhanced green fluorescent protein?eGFP?reporter gene or human sodium-iodide symporter?hNIS?reporter gene were flanked by a couple of AAV-derived inverted terminal repeats?ITRs?,then BV-AAV hybrid viruses?named BIeV and BIhV respectively?were prepared.After infecting rat bone marrow-derived mesenchymal stem cells?rBM-MSCs?and human umbilical cord blood-derived mesenchymal stem cells?hUCB-MSCs?with BIeV,the infection efficiency,eGFP transgene expression level,duration of transgene expression and cytotoxicity/proliferative effects were determined.The characteristics of¹²⁵I^-uptake in BIhV-infected stem cells were investigated by a series of assays in vitro,then the hNIS radionuclide reporter gene imagings were performed after BIhV-infected stem cells transplanting into nude mice.Furthermore,BV was modified with SB100×,and different structural BVs were used to infect tumor cells and long-term transgene expression mediated by SB100×was then tested by comparative assays.Long-term reporter gene imgagings were performed following tansplanting the tumor cells infected by SB100×-modified BV,which also contained eGFP or glucagon-like peptide-1 receptor?GLP-1R?reporter gene?BSeNV or BSGNV?.Moreover,the hUCB-MSCs infected with GLP-1R reporter gene-containing BV-AAV?BIGV?were monitored in vivo by reporter gene imgaging after transplantation.Results:High titers of BV-AAV?all larger than 10⁸ pfu/mL?could be quickly prepared by baculovirus expression system.The in vitro study showed that,the percents of eGFP positive cells?eGFP⁺%?of BIeV-infected rBM-MSCs and hUCB-MSCs were respectively reached 84.25±1.38%and 76.73±3.75%at multiplicity of infection?MOI?=200,which gradually declined to less than 10%in 19 d or 11 d respectively.And there were no cytotoxicities or adverse effects on cell proliferation.BIhV-infected rBM-MSCs and hUCB-MSCs could accumulate ¹²⁵I^-and both reached peak at 30 min in vitro,and the ¹²⁵I^-uptake could be inhibited by NaClO₄.Further correlation assays showed that the BIhV-infected stem cells number significantly correlated with their accumulated ¹²⁵I^-radioactivities?R²=0.9026 and 0.9940 respectively?.Micro-SPECT/CT imagings with ¹²⁵I^-showed that the transplantation sites of BIhV-infected stem cells could be both clearly imaged with high target/background ratios,and the radioactive signals in transplantation sites gradually decreased in 120 min.Moreover,different structural BVs were constructed and prepared,and then used to infected tumor cells?FTC-133 cells and U87 cells?.Comparative assays by flow cytometry showed that the stable tumor cell lines?FTC-133-eN cells and U87-eN cells?which were obtained by SB100×-modified BV infection and drug selection procedure could express eGFP over 180 d.Small animal fluorescence imaging showed that the U87-eN cells in transplantation site could be long-termly monitored in 35 d.And the stable GLP-1R reporter gene-expressing FTC-133 cell line?FTC-133-GN?were transplanted into nude mice,which could be clearly monitored with a high target/background ratio in 50 d by[¹⁸F]AlF-NOTA-MAL-Cys³⁹-exendin-4 image agent and micro-PET imaging,and the radioactive signals in tumor transplantation site didn't decrease in 120 min after image agent administarion.Moreover,an ideal imaging effect with micro-PET was also obtained by imaging the transplanted hUCB-MSCs,which were infected by GLP-1R reporter gene-containing BV-AAV?BIGV?.Conclusion:BV-AAV had high infection efficiency and no cytotoxicity on rBM-MSCs and hUCB-MSCs,and could mediate hNIS reporter gene imaging for monitoring transplanted stem cells,thus there is a feasibility of utilizing BV-AAV-mediated radionuclide reporter gene imaging in stem cells transplantation monitoring.SB100×-modified BV could mediate long-term reporter gene expression,thus allowing long-termly transplanted cells monitorig in vivo.Furthermore,the GLP-1R radionuclide reporter gene imaging had ideal imaging effects,making it a great potential radionuclide reporter gene system.