The Preliminary Study Of Treating Diabetes With Superparamagnetic Iron Oxide Labeled Rabbit Mesenchymal Stem Cells

Background Mesenchymal stem cells(MSCs) can be isolated from bone marrow and so on and can be used cell therapy which have been used cardiovascular diseases,nervous system diseases and so on.Objective The purpose of this study was labeled cells of vitro MRI test and to determine whether SPIO labeled bone marrow MSCs could be detected and tracked noninvasively by MRI in diabetic rabbit models.To explore the labeled efficiency,cellular viability of rabbit mesenchymal stem cells and vitro imaging of the labeled bone marrow MSCs with different concentration and time of superparamagnetic iron oxide(SPIO),and find the best labled concentration and labeled time for clinical vivo magnetic resonance imaging(MRI).We attempt to determine the feasibility of tracing rabbit transplanted cells by using a conventional clinical 1.5T MR scanner.The imaging characteristics and rules of cells also need dynamic observation preliminarily.We hope to find a kind of easy and feasible techniques and methods for future clinical bone marrow MSCs transplant therapy in diabetes.Methods Bone marrow MSCs were derived from bone marrow aspirates of healthy rabbits.Mononuclear cells(MNCs) were isolated using density gradient centrifugation,and cultured and expanded based on plastic adherence.Cellular growth characteristics were observed and recorded under light microscopy.Committed differentiation of bone marrow MSCs into osteoblast by von kossa stain.To identify most cells were bone marrow MSCs,detection and analysis of surface antigen of CD44,CD45 and CD7(?) was performed by flow cytometry.The culture media contained different concentration SPIO paticles were added to the bone marrow MSCs,the ultimate concentration of SPIO was 25μg/ml,50μg/ml,75μg/ml,100μg/ml,150μg/ml respectively.Prussian blue staining,trypan blue exclusion test and vitro magnetic resonance imaging of labeled cells were performed at 24h,72h,1w,2w,3w,4w respectively after labeled, and make sure rational labeled concentration and time.Prepared diabetic rabbit models,transplanted the SPIO labeled bone marrow MSCs and not labeled bone marrow MSCs into diabetic rabbits.Then evaluated the efficience of transplantation by determination of blood sugar and observed the location,distribution and migration.Results(1) the adherenced cells showed spindle shapes and vortex condition growth.Osteogenic induction showed the characters of bone cells.Flow cytometry analysis of the adherenced bone marrow MSCs indicated CD45(-)and CD44(+),CD7(?)(+)demonstrated the cells we get were bone marrow MSCs.(2) The labeled efficiency of all the groups reached to 95%-100%.trypan blue exclusion test showed the viability of the labeled cells with SPIO of 0μg/ml and 25μg/ml,0μg/ml and 50μg/ml,25μg/ml and 50μg/ml was not significantly statistical difference(P>0.05).The viability of the cells labeled with SPIO of labeled cells of ld and 3d was not significantly statistiscal difference(P>0.05).Vitro MRI test showed when labeled time is 72h and labeled concentration is 50μg/ml,signal intensity change is most remarkable.(3)The blood sugar of diabetic rabbits models are all more than 16.8mmol/L.Determination of blood sugar showed bone marrow MSCs transplantation can decrease the level of diabetic rabbits' blood sugar. SPIO labeled cells could be identified on T2WI and T2~*WI sequence after injection into both normal and diabetic rabbits by using 1.5T MR.Bone marrow MSCs were observable and could be seen for at least 10days.Conclusion Bone marrow MSCs is easy to be isolated and purified by combined using density gradient centrifugation and plastic adherence.The SPIO concentration of 50μg/ml and the labeled time of 72h not only have high labeled efficiency and no effect on the viability of bone marrow MSCs but also signal intensity change is most remarkable with MRI in vitro,can be considered optimal labeled concentration and time with SPIO.Labeled bone marrow MSCs and non-labeled bone marrow MSCs can cure the hyperglycemia of the diabetes.Visualization of SPIO-labeled bone marrow MSCs in diabetic rabbit models by using a conventional 1.5T MR is feasible and the ability may be used to future clinical studies.