| Background and objectiveTracking stem cells in vivo is a hot topic in the research field of molecular imaging. Two methods for tracing transplanted cells have been applied: direct magnetically labeling and genetic reporter imaging, in which the latter has more advantages. Ferritin(FTH1) as a mature MRI reporter gene has been successfully used for tracking many types of cells. At present, most studies showed that, though expression of FTH1 is not affected by cells proliferation, whether it is affected by differentiation of stem cells is still unclear. In this study, we established the bone marrow mesenchymal stem cells(MSCs))carrying FTH1 gene under the control of a Tet-On switch(MSCs-Tet-FTH1) by reconstructing the lentiviral vector and infect the, MSCs, and induced the MSCs-Tet-FTH1 to differentiate into neuron-like cells. The FTH1 expression in the MSCs before and after differentiating into neuron-like cells were detected. Our aim was to explore whether the stem cell differentiation has a impact on the expression of gene FTH1, and whether the capacity of gathering iron in differentiated cells carrying FTH1 can lead to enough MRI signal change, ultimately supply an in vivo, safe and long term MRI tracking method for implanted stem cells as well as the differentiated neuron like cells.Methods1 Construction of lentiviral vector carrying FTH1 gene regulated by Tet-on and virus packagingThe c DNA sequence of FTH1 was obtained from the gene bank. By PCR and sequencing DNA recombinant, the gene was inserted into the Tet-on inducible lentiviral vector. Then 293 T cells were co-transfected with the reconstructed lentivirus vector plasmid and lentivirus packaging plasmids to produce the lentivirus LV-Tet-FTH1. ELISA technology was used to detect the titer of the lentivirus.2 Transfecting cells with lentivirus carrying FTH1 and screening successfully transfected cellsThe MSCs were successfully transduced with lentivirus LV-Tet-FTH1, and screened by using puromycin..3 Detecting the gene expression product ferritin and its capacity of gathering ironAfter FTH1 gene carried by lentivirus vector were inserted into the host cell genome, the expression of gene need to be started by the inductive agent doxycycline(DOX). By adding a concentration of DOX to complete medium to culture MSCs-Tet-FTH1, the expression of FTH1 peaked after a certain time, Western blot was used to detect the gene expression product FTH1 to determine the optimal concentration of DOX and the optimal expression time. CCK-8 reagent testing was performed to test whether the proliferative activity of MSCs was affected by transfecting virus or/and ferric ammonium citrate(FAC). Prussian blue staining and electron microscopy were used to observe the cells capacity of gathering iron, and a 3.0 T MRI was performed to observe the signal changes in status of gene expressing and gene silencing.4 Detecting specific markers of neuron-like cells which was differentiated from MSCsAll-trans retinoic acid and MNM neural induction was used to induce cells into neurons-like cells. To determine whether the MSCs have differentiated into the neuron-like cells, the cells morphological changes were observed, and the neuron-specific surface markers, including neuron-specific enolase(NSE), nestin(Nestin) and microtubule-associated protein-2(MAP-2), were identified.5 Detecting the gene expression product of FTH1 and its ability to gather iron after differentiating into neurons-like cellsWhen MSCs differentiated into neuron-like cells, Western Blot was used to detect the gene expression products ferritin. Prussian blue staining and transmission electron microscope was used to detect the capacity of iron gathering, and a 3.0 T MRI was performed to found the signal changes of T2 WI in FTH1-Tet-MSCs.Results1 Construction of lentiviral vector carrying FTH1 gene which can be regulated by Tet-on, packaging virus and determining its titerUsing DNA sequencing, gene recombination technology demonstrate Tet-on regulated lentiviral vector system carrying FTH1 gene has been successfully constructed, determinning the titer of virus was 1×109 Tf U / ml.2 Tranfecting MSCs with lentiviral vector carrying FTH1 of MSCs and screening successfully transfected cells and detecting its capacity of gather ironThe MSCs carrying the reporter gene FTH1 under the control of a Tet-On switch(MSCs-Tet-FTH1) were successfully established.3 Inducing gene FTH1 carried by lentiviral vector with a Tet-on switch to express by DOX and detecting its capacity of gathering ironWestern Blot results showed that, gene expression of FTH1 was closely dependant on the dose and duration of DOX supplementation. With 1μg/ml of DOX inducing, gene expression product of FTH1 peaked at 3th day.By adding a certain concentration of DOX and ferric ammonium citrate(FAC) in a complete medium, the cells can utake iron. 3.0 T MRI scanning showed: signal in MSCs-Tet-FTH1 cells group was significantly reduced, while the control group showed no signal change. Prussian blue staining and transmission electron microscope showed a large number of iron particles in MSCs-Tet-FTH1 group after adding 1μg / ml DOX and 500μM FAC. While the control group MSCs showed no particle on Prussian blue staining and transmission electron microscope.4 MSCs-Tet-FTH1 differentiating into neuron-like cellsAfter MSCs were induced by all trans retinoic acid(ATRA) and MNM for 24 h, the cells morphology changed significantly, showing the typical neuronal morphology. Specific markers NSE, Nestin, and MAP-2 expression was strongly detected. In the experimental group cells, MSCs-Tet-FTH1 also were detected with these changes. This means transfecting virus had no effect on the of differentiatiing ability of MSCs.5 FTH1 expression and its ability to gathering iron after differentiating into neurons-like cellsAfter MSCs-Tet-FTH1 was induced to differentiate into neuron-like cells, Western Blot results showed Neurons-Tet-FTH1 can still express ferritin; Prussian blue staining discovered a large number of blue-stained iron particles in the cells, transmission electron microscope also detected there are a lot of black dense granules. A 3.0 T MRI detected significant decrease of T2 WI signal of Neurons-Tet-FTH1.ConclusionWe successfully constructed lentivirus vectors carrying FTH1 with a Tet-on switch, and infected MSCs, and demonstrated that the FTH1 gene expression was induced by DOX agent with a closely dose- and time-dependant manner. The MSCs-Tet-FTH1 was successfully induced to differentiated into neuron-like cells. The expression of gene FTH1 had no significant impact on cells diffrentiation, and the cells differentiation did not affect the expression of gene FTH1. MRI based on reporter gene FTH1 can be used to tracking MSCs differentiating into neurons in vitro, thus provides a incucible, secure and long-term tracking method forstem cells. |
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