Research on biological activities and MRI of differentiations of neural-like cells induced by superparamagnetic iron oxide and green fluorescent protein double-labeled bone marrow-derived mesenchymal stem cells in vitro

Abstract

Objective To explore the biological activities and the MR imaging signal intensities(SIs) characteristics of differentiations of neural-like cells induced by superparamagnetic iron oxide (SPIO) and green fluorescent protein (GFP) double-labeled bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. Methods GFP-BMSCs were labeled with different concentrations of SPIO in vitro (the concentration of the A, B, C and D group was 25, 50, 75 and 100 ug/ml, respectively; the E group without labels of SPIO served as the control group). The Prussian blue stainings were used to detect the labeling rates of SPIO. The iron contents of cells were measured by atomic absorption spectrometer. The CCK8 experiments were used to detect the cell proliferation rates. The cell cycles were detect by PCR. Each of the A- E groups had a test tube with 1 × 108 cells.All test tubes underwent T2* weighted imaging (T2*WI)and susceptibility weighted imaging (SWI) in a MR imaging scanner. The optimal group was defined by comparing the measurements of SIs between T2*WI and SWI. The optimal group and the E group together induced the differentiations of osteogenesis and neural-like cells. The stainings of alizarin red, osteocalcin and Nissl, NeuN, and NF-200 were performed at 72 hours. Real-time quantitative PCR was used to detect the expression levels of RNA in tub3, nestin, NSE, MAP-2 and Syt1. The positive staining rates and the expression levels of RNA were compared between the two groups. Finally, SWI was used to analyse the changes of SIs. One-way analysis of variance (ANOVA) was used to the multi-group comparison. Using least significant difference (LSD) test to analyse the comparisons between the multi-groups. Results The labeling rates of the A- D groups were 100%. The iron contents of cells in the A- E groups were (14.36 ± 7.61), (21.73±3.42), (30.54±8.73), (33.65±9.62), and (2.31±0.32) pg/cell, respectively. The iron contents of cells in the A-D groups were significantly higher than those in the E group (F=3.852,P=0.003). There was no significant difference between the C and D groups (P=0.267). In all groups, the D group had the lowest OD value in the CCK-8 experiments (3.18 ± 0.46). In the A- E groups, the changes of SIs in SWI were significantly lower than those in T2* WI.There was no significant difference in SIs of SWI between the C group (145.89±14.31) and the D group (127.37±12.21). Except the comparison between the group C and D, the comparisons between all the groups had significant differences (P 0.05). The expression levels of tub3, nestin and NSE were significantly higher before than after induced differentiations(P<0.01). SIs of SWI had no significant difference between before and after induced differentiations in the C group (t=1.26,P=0.236). Conclusions SPIO and GFP double-labeled BMSCs can induce neural-like cells without influencing biologic activities. MR SIs are decreased by the increase of SPIO concentrations in cells. SWI was the most sensitive sequence. The SIs of SWI has no differnce between before and after induced differentiations. Key words: Mesenchymal stem cell; Magnetic resonance imaging; Molecular probe