The thrombogenic risk induced by intraportal infusion of Adult Derived Human Liver Stem/Mesenchymal Cells in Wistar rats can be controlled with a combination of anticoagulant drugs, heparin and bivalirudin.
Intro : Mesenchymal Stem cell (MSC) transplantation is a fast emerging therapy for regenerative medicine. However MSCs express a procoagulant activity (PCA) inducing a thrombogenic risk in recipient patients. This thrombogenic risk is due to activation of the coagulation cascade, triggered by Tissue Factor (TF) on MSCs, resulting in fibrin production and platelet consumption. In vitro the PCA of Adult Derived Human Liver Stem/Mesenchymal Cells (ADHLSCs) can be controlled by an anticoagulant cocktail, combining an antithrombin activator (Heparin) and a thrombin inhibitor (Bivalirudin). Aim : The aim was to study in vivo, the effect of an anticoagulant cocktail on thrombogenic risk induced by intraportal ADHLSCs. First we wanted to characterize the thrombogenic events induced by ADHLSCs infusion. Then we wanted to study the effect of the anticoagulant cocktail on these events. Finally the effect of cell dosage on the thrombogenic risk was studied. Method : Wistar rats (n=3 per group) were infused with ADHLSCs through an intraportal catheter. Rats were infused with 3 different cell dosages (50, 12.5 and 5 million ADHLSCs/kg), with or without administration of anticoagulant therapy (heparin and bivalirudin). Control group was infused with PBS. Blood samples were collected before and 1 hour after cell infusion. Hemoglobin and platelet levels were analysed with an automated hematology analyzer, Cell-Dynn Emerald (Abbott). Rats were sacrified and liver tissue was collected 1 hour after cell infusion. Serial slides were performed to analyse the localisation of AHDLSCs (human Beta 1 integrin staining) and fibrin (P.T.A.H. coloration). Cell quantification and localisation was analysed with digital imaging system, Visiopharm. Fibrin was analysed in Portal Veins (PV) containing ADHLSCs by digital imaging system, TissueIA. Result : ADHLSCs intraportal infusion induced after 1 hour a significant decrease (p < 0.05) in platelet count with production of fibrin around the infused cells localized in PVs. No platelet consumption or fibrin production was observed in the control (PBS) group. In the anticoagulated group, platelets also decreased significantly (p < 0.05) but fibrin production was significantly (p < 0.001) reduced, from 86.6% ± 6.4 to 26.4% ± 13.8. When different cell dosages were infused, we observed a correlation between the number of infused cells and the number of cells in the liver. The number of cells in liver tissue (cells/mm2) and the number of PVs containing cells (PVs containing cells/total PV) were significantly (p < 0.01) lower when lower cell dosages were used, compared to the higher cell dosage group. Consumption of platelets was also correlated to cell dosage. No decrease in platelets was observed when 5x106cells/kg were infused without anticoagulation, and when 12.5x106cells/kg were infused with anticoagulation. Platelets tended to decrease less in the high cell dose group, 50x106cells/kg when anticoagulant therapy was administered. No significant difference has been observed in the production of fibrin in PVs between the different cell dosages. In all conditions, hemoglobin did not decrease significantly, meaning that blood sampling did not interfere with our results. Conclusion : Intraportal infusion of ADHLSCs activated the coagulation cascade resulting in fibrin formation and platelet consumption 1hour after cell infusion. The use of a combination of anticoagulant drugs, heparin and bivalirudin, could prevent the formation of fibrin, but a significant decrease in platelets was still observed. However this decrease tends to be less and can even be controlled when lower cell dosages are used. The production of fibrin and thus the activation of the coagulation cascade was not dependent of cell dosage
