The Experimental Research On The Comparison Of Three Methods Labeling Tibetan Miniature Pigs MSCs And The Distribution Of The Labeled MSCs In Allografted Kidney

Part Ⅰ The comparison of three methods to label Bone marrowMesenchymal Stem Cells of Tibetan miniature pig with SPIO, CM-DiIand EGFP in vitroObjective: To investigate that using SPIO, CM-DiI and Lentivirus vector carrying EGFPgene labeling Bone marrow Mesenchymal Stem Cells of Tibetan miniature pig in vitro,and to provide an experimental supporting for further study of tracing MSCs aftertransplantation in vivo.Methods: Tibetan miniature pig’ MSCs were isolated and cultured using the densitygradient centrifugation and adherent method, and then were identified by differentiatedcapability and special cluster of differentiation on the cells surface. The third passageMSCs were labeled with SPIO、CM-DiI and Lentivirus vector carrying EGFP gene,respectively. Viability evaluated by trypan blue exclusion. And the suitable labeledcondition of the three methods explored which didn’t influence the cells’ biologicalcharacteristics via MTT results. The cells’ labeling efficiency on different passages wereobserved using fluorescence microscopy or Fluorescence activated cell sorting（FACS).Results: The cultured cells were accord with the bio-characteristics of MSCs byinduction differentiated into adipogenic, osteogenic and the detection of the cell surfacemolecules. The third passage of MSCs were incubated and labeled using differentconcentration of SPIO (12.5ug/ml,25ug/ml, and50ug/ml). Following Prussian bluestaining, the cytoplasm of MSCs contained numerous blue particles. The labelingefficiency reached to100%. From the passage1to2, blue-stained particles graduallyreduced in cytoplasm. Concentration of25ug/ml was the optimal for labeling MSCs and had no impact on the viability and proliferation of MSCs at different time points afterlabeled. Under the fluorescent microscope, the labeled cells by different concentration(2umol/ml、5umol/ml、8umol/ml、10umol/ml) of CM-DiI gave out red to orange light.It was no significant impact that the concentration of10umol/ml CM-DiI was found onthe cells morphology and growth. Using FACS analysis, the labeling rate were98%afterthe first of labeled. With the labeling time pasted, the labeling rates from P1to P2were52.9%,14.2%, respectively. And the fluorescence intensity decreased gradually.Lentiviruses carrying EGFP gene successfully infected MSCs and then produced EGFP.Bright green fluorescence transfected cells could be observed under fluorescencemicroscopy after96h of transfection. FACS for determining the EGFP expressionefficiency was83.9%, and the green fluorescent intensity without remarkably weakenedwith the cocultured time lasted. The labeling rate of P1and P2were84.1%,84.6%,respectively. It was no impact on the cells proliferation of virus transfection.Conclusion: SPIO, Fluorescein based dye CM-DiI and lentivirus-mediated EGFP genetransfer can label bone marrow mesenchymal stem cells of Tibetan miniature pig in vitro.Three methods compared with each others, SPIO, CM-DiI were very effective method tolabel MSCs. But the labeling stability was relatively worse; they may be suitable toshort-tern labeling within two weeks. While lentiviruses carrying EGFP gene labelingmay be more stable for long-term tracing MSCs. Part Ⅱ The experimental research on the distribution of labeledTibetan miniature pig MSCs in allografted Kidney injected byallografted renal arteryObjective: To Confirm the feasibility of tracking injected MSCs labeled by SPIO andCM-DiI in vivo and to observe the distribution in allografted kidney of Tibetan miniaturepig’ MSCs injected by allografted renal artery at the vessels reconstruction.Methods: Tibetan miniature pigs were selected to establish allogeneic renal transplantation models. Autologous MSCs labeled by SPIO or CM-DiI injected byallografted renal artery. The recipients were divided into three groups according to theinjecting or not injecting and labeling methods of MSCs. Group A: control, no injectedMSCs (n=2); Group B: injected SPIO-labeled MSCs (n=2,2×106/kg); Group C: injectedCM-DiI-labeled MSCs（n=2,2×106/kg). After the recipients executed, the renalallografts paraffin-cut and frozen sections were made to observe the labeled MSCsdistribution by an optical or fluorescence microscope.Results: We have established6cases Tibetan miniature pigs’ renal transplantationmodels and injected autologous MSCs via allografted renal artery. The allografts werenephrectomized after24hours.One case was found diffuse hemorrhage in bothglomerulus and renal interstitial that indicated allografts rejection by Banff2007criteria.Prussian blue staining of allografted kidneys with MSCs injected show that most of theinjected MSCs was in an irregular shape with intracytoplasmic blue particles located inthe renal glomeruli. The amounts of blue particles located in glomeruli were more than inrenal interstitial per section(2.0±1.7vs1.2±1.1，p<0.05). The frozen section of kidneycortex and medulla under fluorescent microscope both observed red fluorescent points,and there were more labeled MSCs in renal cortex(3.2±2.4) than in renal medulla(2.0±1.6) by Statistical analysis(p<0.05).Conclusion: SPIO and CM-DiI labeling can be used to observe injected MSCs byallografted renal artery after the vessels reconstruction. SPIO labeled MSCs injected byallografted renal artery observed mainly distributing in the glomerulus; CM-DiI labeledMSCs were found at the cortex department of the allgrafted kindey.