NSE-Promoter Initiating FTH1 Reporter Gene Specific Expression And In Vitro Magnetic Resonance Imaging In Neural Differentiation Of The Bone Marrow Mesenchymal Stem Cells

Background and objectiveMagnetic resonance imaging(MRI)has become one of the important methods for transplanting stem cells due to its high spatial resolution,well soft tissue contrast,and lack of ionizing radiation.At present,there are mainly two methods for tracing the transplanted cells using MRI,namely direct imaging through magnetic labeling and indirect imaging through reporter gene expression.However,the direct labeling method has an inherent defect that the intracellular labeling markers will continue to degrade as the cells divide and proliferate,failing to achieve long-term traceability of the transplanted cells.Reporter gene based MRI uses genes that are continuously and stably expressed in cells to long-term trace the transplanted cells and overcome the limitation of direct imaging of magnetic markers.Among the many reporter genes used for MRI,the ferritin gene is the most widely studied.The endogenous ferritin is commonly found in organisms and consists of a protein shell and an iron core.The protein shell is surrounded by two subunits:the heavy chain and light chain.The heavy chain polypeptide 1(FTH1)has the activity of ferroferric oxidase,and can induce the expression of transferrin receptor and promote iron absorption,thereby causes changes in MRI signals.As a functional magnetic resonance imaging report gene,it has become one of the hotspots in molecular imaging studies.So far,there have been many studies using the FTH1 reporter to achieve continuous tracing of a variety of cells.In our previous studies,we used lentiviral vector to transfer FTH1 reporter gene into bone marrow mesenchymal stem cells(MSCs),and induced the differentiation of transgenic MSCs into neuron-like cells.We found that although the induced cells changed in morphology and the specific markers,there was no significant difference in the expression of FTH1 between MSCs and neuron-like cells.As a result,no MRI signal change was found in cells before and after neural differentiation.These results showed that the use of MRI reporter gene imaging alone can not distinguish between the two cells before and after neural differentiation,that is,this imaging method can not be used to determine the occurrence of stem cell differentiation events.The key to solve this problem lies in finding a gene that specifically and highly expresses in nervous tissue cells.Using this gene as a promoter to drive the expression of MRI reporter gene can theoretically monitor the neural differentiation of stem cells through MRI.In this study,a lentivirus carrying the reporter gene FTH1 driven by a neuron-specific enolase(NSE)promoter were constructed,and transfected into the MSCs.In the differentiation of the transgenic MSCS into neuron-like cells,MRI was performed to observe the signal change.Our aim was to explore the feasiblily of using MRI to detect the neurally differentiated cells through the specific expression of the reporter gene FTH1 driven by an NSE promoter,and lay the foundation for further MRI to monitor neural differentiation of transplanted stem cells.Methods1 Construction of the lentiviral vector and the virus packagingThe cDNA sequence of NSE promoter was artificially synthesized and ligated with FTH1 gene sequence.The PCR product and vector LV5 were digested,ligated and transformed to obtain the recombinant lentiviral vector plasmid LV5-NSE-FTH1-GFP-Puro.293T cells were transfected to produce the virus particles.The viruses were collected after 48 h,named NSE-FTH1.The virus titer was determined using a per well-dilution titer assay.the NSE-LV was constructed as a control.2 Transfection of MSCs with recombinant lentivirusMSCs were transfected with NSE-FTH1.The fluorescence expression of the cells was observed under the microscope 48 hours later.Puromycin was selected to obtain the stable cell line experimental group and the control group was established.3 Neural differentiation and identification of MSCs in vitroThe cells were seeded in 6-well culture plates and pre-incubated with 1mmol/L ATRA for 24 h,then induced with MNM neural induction for 24 h.The morphology of the induced neuron-like cells was observed under a microscope.The expression of the neuron-specific surface markers,including NSE,nestin,and microtubule-associated protein-2(MAP-2)were detected by the immunofluorescence.4 FTH1 expression and iron collection before and after neural differentiationThe expression of FTH1 gene in MSCs was detected by Western blot.The distribution of iron particles in each group was observed by Prussian blue staining and transmission electron microscope.The CCK-8 reagent wes used to detect the cell proliferation activity after infection with the lentivirus of the reporter gene or/and with the addition of FAC.hematoxylin-eosin(HE)staining was used to observe the morphological changes in the cells.5 MRI before and after neural differentiationThe spin echo(SE)T2WI scan was performed on a 3.0T MRI machine and the MRI signal changes were observed before and after neural differentiation of MSCs.Results1 Construction and identification of NSE-FTH1 recombinantlentivirus vector.The recombinant lentiviral vector(LV5-NSE-FTH1-GFP-Puro)was successfully constructed by gene recombination,PCR,and other techniques.The lentivirus was produced with a titer of approximately 3x10~8 TU/ml which met the experimental requirements.2 Transfection of MSCs with recombinant lentivirusThe MSCs were successfully transfected with recombinant lentivirus,named MSCs-NSE-FTH1,and they were screened by puromycin.3 Neural differentiation of MSCsAfter neural induction,the MSCs differentiated into the neuron-like cells,which presented the morphology of neurons.Immunofluorescence detected obvious expression of NSE,nestin,and MAP-2 in each group of neuron-like cells.However,there was no significant expression of these markers in the MSCs.4 FTH1 expression and iron collecting effect before and afterneural differentiationWestern blot analysis showed that the expression of ferritin increased significantly after the MSCs-NSE-FTH1 cells differentiated into neuron-NSE-FTH1 cells,and the difference was statistically significant(P<0.01).There was no significant difference in the expression level of CMV promoter groups(P>0.05).Both Prussian blue staining and transmission electron microscopy confirmed that more iron particles accumulated in the Neurons-NSE-FTH1 group.The results of CCK-8 reagent showed that the cell proliferation activity was decreased after infection with the lentivirus carrying the target gene or/and culture.HE staining showed that in all groups of cells,there was no abnormal change in cell morphology under the expression of FTH1 and the accumulation.of iron.5 MRI findings in each group of cells3.0T MRI scan showed that T2WI signal was significantly reduced after MSCs-NSE-FTH1 was induced to neural differentiation.However,in the CMV promoter group,T2WI signal did not change after neural differentiation.ConclusionThe expression of the reporter gene FHT1 can be triggered by the neuron-specific NSE promoter in the neural differentiation of MSCs.MRI based on the reporter gene FTH1 driven by NSE could be used to monitor the occurence of neural differentiation event.This study lay a foundament for in vivo MRI tracing of neural differentiation of transplanted MSCs.