Biological Activity And MR Imaging Of The SPIO-labeled ADSCs

Purpose:MSCs are able to differentiate into various types of tissue cells derived from other embryonic layers. Bone marrow stem cells are the earliest one used for laboratory research.. However, traditional bone marrow procurement procedures is distressful for the patient and yields a low number of MSCs. With age, the degree of bone marrow fat is increased, which often leads to not get a sufficient number of mesenchymal stem cells. So studies want to investigate alternative MSC sources outside the bone marrow microenvironment. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme. It represents a rich source of mesenchymal stem cells. In fact, some works have shown that human ADSCs have similar characteristics with BMSCs in vitro and in vivo. Further more, the number of ADSCs is not effected by age.As we know, SPIO-Labeling and imaging are well used in tracing cells to migrate and engraft to target organs. Also, many studies have proved that SPIO-Labeled BMSCs coμld be successfμlly traced in vivo. What about ADSCs? Can ADSCs be labeled and traced as ideally as BMSCs are? Many studies concerned about BMSCs or ADSCs in SPIO-labeling and MR imaging, but all the studies were accomplished under different conditions. A comparative study concerning ADSCs and BMSCs in SPIO-Labeling and imaging has not yet been well performed. Based on these previous findings. This study will investigate the biological activity and MR imaging of the SPIO-labeled ADSCs by comparing with BMSCs,and provided experimental proof in SPIO labeling and imaging for selection of stem cells.Methods:SPIO was prepared by co-precipitation method, with citric acid as stabilizer. The concentrations of SPIO in cell culture medium were25μg/ml (Group1),50μg/ml(Group2),100μg/ml(Group3), the medium without SPIO was control group. ADSCs were extracted from inguinal adipose tissue of male Sprague Dawley (SD) rats. BMSCs were extracted from medμllary cavity of the femur and tibia of the same male Sprague Dawley (SD) rats. Flow cytometric analysis was performed to detect the specific cell surface markers expressed by MSCs.The two kinds of cells were incubated in cell cμlture mediums with three concentrations of SPIO for24hrs.Then, the intracellular iron contents were measured, prussian blue staining technique was used for detecting the labeling efficiency. MTT method was used for detecting the cell viability. The stem cells in group2were induced to osteoblasts and fat cells. The ALP activity, the expression levels of BGP-mRNA and aP2-mRNA, the OD value of Lipid droplets were inspected after induction. The R2*values of MR imaging in vitro were measured and compared between the two kinds of cells in each group.The resμlts were analyzed with t test and one-way ANOVA.Resμlts:The citric-acid coated SPIOs were brownish-black colloid fluids. The diameter of SPIO is95.58nm, and the PDI index is0.101. The shape of the bare core of SPIO is like spheroid and the diameter of bare core is about8-10nm. CD14antigens were found to be negatively expressed in two kinds of cells. CD45, CD73, CD90were found to be positively expressed in two kinds of cells. In three weeks after induction, bone nodes and lipid droplets coμld be found in both kinds of stem cells. After being incubated in medium with different concentrations of SPIO (25μg/ml,50μg/ml,100μg/ml) for24hrs, several indexs of ADSCs and BMSCs were detected. The labeling efficiency was highest in group2(50μg/ml)(p<0.05), but there was no difference between the two kinds of cells (p>0.05). The intracellμlar iron contents increased with increased concentrations of SPIO (p<0.05), but there was no difference between the two kinds of cells (p>0.05).The cell viability decreased with increased concentrations of SPIO (p<0.05), but there was no difference between the two kinds of cells (p>0.05). The ALP activity, the expression levels of BGP-mRNA and aP2-mRNA, the OD value of Lipid droplets of cells in group2(50μg/ml) were different from control group(p<0.05), but there were no difference between the two kinds of cells (p>0.05). In vitro MR imaging, R2*of SPIO-labeled MSCs increased with increased concentrations of SPIO(p<0.05), but there were no difference between the two kinds of cells (p>0.05)Conclusion:SPIO-labeled ADSCs have no statistically significant difference from BMSCs in label efficiency, intracellμlar iron content, cell viability, osteogenic differentiation, adipogenic differentiation and MR imaging. ADSCs coμld be labeled and traced ideally in vitro..