Novel triple fusion reporter gene for labeling and tracking human mesenchymal stem cells in vitro

Abstract

Objective To evaluate the feasibility of labeling and tracing human bone marrow mesenchymal stem cells (hMSC) by a triple fusion reporter gene imaging.Methods A lentivirus vector,the pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP,which contained optical reporter gene-enhanced green fluorescent protein (EGFP),bioluminescent reporter gene-luciferase2 and MR reporter gene-ferritin,was constructed by gateway technology.After the accuracy of gene sequence was validated,the lentivirus was packaged and infected hMSC,and a stable transformed hMSC cell line was established by fluorescence-activated cell sorter.The expression of EGFP was inspected with fluoresence microscope; the bioluminescence photon number was counted by optical imaging system in 1 s after D-luciferin was given; the iron content of the cells was detected by prussian blue iron stain; T2 values of the T2-mapping of MRI were measured.After an extended series of 3 to 5 times repeated experiment,results were compared between transfected hMSC and un-transfected hMSC to explore the efficiency of the labeling and tracking hMSC of the reporter genes.The positive rate of expression of the EGFP,the difference in iron stain and MR signal (T2) between the two groups was measured by t test.The bioluminescence photon detected in two gradient groups was count to set a Pearson correlation analysis.Results The gene sequence of pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP was confirmed by DNA sequencing analysis.The lentivirus vector was successfully constructed and infected hMSC.A stable transformed hMSC cell line,with triple fusion reporter genes,was successfully established for the follow-up test.Single hMSC expressed EGFP could be detected by fluorescence microscope.The optical signal of 2.00× 106,1.00× 106,5.00× 105,2.50× 105,1.25 × 102,6.25 × 104/ml cells could be detected as 7.972× 107,3.061 × 107,1.547 × 107,7.805 × 106,4.256 × 106,1.411 × 106 bioluminescence photon after D-luciferin was given,and there was linear positive correlation between the intensity of optical signal and the number of cells (r=0.993,P＜0.01),while no optical signal was captured under the same experimental condition.The iron stain rate of transfection cells was (81.6± 5.1)％,which was significantly higher than that of un-transfection cells (21.6±3.6)％ (t=20.97,P＜0.01).MR showed the difference of T2 value of 2.5× 106 cells per milliliter between the transfection (628.0±23.1)ms andun-transfection cells (646.5±17.2)ms,but the difference was not significant (t=1.286,P=0.246).However,the difference was obvious when ammonium ferric citrate was given,(488.3± 10.7)ms in the transfeetion cells and (520.8 ± 21.0)ms in the un-transfected cells (t=2.76,P=0.033).Conclusion A triple fusion reporter gene imaging,including EGFP,luciferase2 and ferritin,is feasible,which can successfully be transfected into hMSC and effectively expressed in the daughter cells. Key words: Human bone marrow mesenchymal stem cells ; Gene imaging; Comparative study