MRI Study Of MSCs Labeled With SPIO And Fth1 Gene Differentiation Into Cardiomyocytes In Vitro

Objective The purpose of this study was to evaluate the feasibility, efficacy and safety of rat bone marrow mesenchymal stem cells(MSCs) labeled with ferritin gene Fth1. To observe the effects of different concentrations of superparamagnetic iron oxide(SPIO)and Fth1 marker gene on proliferative activity of MSCs. To discuss the influence of SPIO and Fth1 gene on MSCs differentiation into cardiomyocytes. To study the MRI findings in vitro of MSCs labeled with SPIO and Fth1 gene differentiation into myocardial cells and differences between them.Methods 1. This study used whole bone marrow adherent directly to isolate and culture SD rat MSCs, flow cytometry to detect cell surface antigens CD34, CD44 and CD90,osteogenic and adipogenic induction of differentiation to identify the MSCs.2. To construct a ferritin heavy chain gene Fth1 lentiviral expression vector. Fth1 reporter gene lentivirus transfected MSCs and fluorescence microscopy observed EGFP(enhanced green fluorescent protein) expression, q RT-PCR and Western blot detected Fth1 gene and protein expression.3. Different concentrations(25μg/ml, 50μg/ml, 75μg/ml, 100μg/ml) of superparamagnetic iron oxide(SPIO) labeled MSCs; Prussian blue staining detected SPIO labeling efficiency; CCK-8 assayed SPIO-labeled MSCs proliferative activity.4. Use different concentrations of ferric citrate medium(0.1 mol / L, 1 mol / L, 3 mol /L, 5 mol / L, 7 mol / L) cultured Fth1-MSCs, Prussian blue staining detected cells iron uptake situation, CCK-8 assayed proliferation of Fth1-MSCs.5. 50μg / ml SPIO and Fth1 labeled MSCs in vitro by 10μmol / L 5-aza induced differentiation into cardiomyocytes, after 28 days pathology staining detected the expression of cardiac specific markers(a-actin and cardiac troponin T). At different time points after labeled, MSCs labeled with SPIO and Fth1 differentiated into cardiomyocytes was detected by MRI, to observe the changes of signals and two sets of signal differences.Results 1. MSCs from whole bone marrow adherent directly were spindle-shaped,radially arranged and adherent growth, CD34、CD44 and CD90 positive cells rates were2.8%, 99.8%, 99.5%, with differentiation to bone cells and adipocytes ability.2. By restriction enzyme digestion of PCR and recombinant vector gene sequencing, we successfully constructed overexpression lentivirus vector p CDH-EGFP-Fth1 that carry ferritin heavy chain Fth1 and fluorescent protein gene.3. Fth1 gene was successfully transfected MSCs, observed by fluorescence microscope about 80% MSCs expressing EGFP, q RT-PCR and Western blot detection of ferritin heavy chain gene and protein expression increased significantly compared with ordinary MSCs.4. Compared with 25 μg / ml, 75μg / ml and 100μg / ml, the concentration of 50 μg / ml of SPIO labeled MSCs had high efficiency, up to 98%, and had a small effect on the proliferation of MSCs, and compared with the concentration of 25 μg / ml group, the difference was not statistically significant(P>0.05). However, Although the concentration of 75μg / ml and 100μg / ml of SPIO had the high labeling efficiency, but they had a significant effect on the proliferation of MSCs, compared with the control group, 25 μg / ml group and 50 μg / ml group differences were statistically significant(P<0.05).5. No added iron citrate culture medium, Fth1-MSCs proliferation and the control group showed no significant difference(P> 0.05); Added at a Fe concentration of 0.1 mol / L of iron-containing medium, Prussian blue staining was not observed blue granules; Each Fth1-MSCs cytoplasm had blue granules when added at a Fe concentration of 1 mol / L,3 mol / L, 5 mol / L, 7 mol / L of conditioned medium, and with the increase of the iron concentration of conditioned medium, the staining gradually deepened. But with increasing concentration, cell morphology was affected, and compared with control group, the cell proliferation differences were statistically significant(P <0.05).6. 50 μg / ml SPIO and Fth1 labeled MSCs differentiation into cardiomyocytes could both express cardiac-specific markers(a-actin and cardiac troponin T). With SPIO concentration increased, MRI of T2 signal decreased; SPIO-labeled MSCs during differentiation into myocardial cells, signal gradually reduced over time to 28 days without reducing on MRI T2 signal, the same as the previous mark. After added iron citrate medium 5 days MRI T2 signal of Fth1-MSCs was reduced, and the signal had no significant change with time, 28 days MRI T2 signal compared with 5 days and 14 days,there was no significant difference(P> 0.05).Conclusion 1. We can successfully construct Fth1 lentiviral overexpression vector and transfected Fth1-MSCs could stably overexpress Fth1 gene, does not affect its proliferation, but there was a certain influence on cell proliferation after adding ferric citrate medium.2. 50 μg / ml concentration of SPIO efficiently labeled MSCs, MRI signal changes significantly, and the effect on cell proliferation is relatively small, could be the optimal SPIO labeled concentration of MSCs.3. Fth1 lentivirus overexpressed has no effect on Fth1-MSCs proliferation with no added iron citrate culture medium, and the higher the conditioned medium iron concentration, the more cellular uptake of iron, but the greater effect on cell morphology and proliferation, so we choose 1 mol / L ferric citrate medium is more appropriate.4. 50 μg / ml concentration of SPIO and Fth1 reporter gene marker, did not affect the ability of MSCs differentiation into cardiomyocytes. MR can detect signal change of 50μg / ml concentration SPIO and Fth1 labeled MSCs during differentiation into cardiomyocytes, 50 μg / ml SPIO labeled MSCs duringdifferentiation into cardiomyocytes low signal gradually increased with time, after 28 days the low signal disappeared, while cardiac cell differentiation of Fth1-MSCs with the addition of conditioned medium after 28 days the signal did not change significantly, can be the long-term monitoring of the tracer.