Intraportal infusion of human liver-mesenchymal stem cells in rats lead to transient interruption of the hepatic blood flow: intravital microscopy and anapathological analysis

Previous studies showed that liver Mesenchymal Stem Cells (MSC) express a procoagulant activity (PCA) that can be controlled in vitro by an anticoagulant cocktail, combining an antithrombin activator (Heparin) and a thrombin inhibitor (Bivalirudin). First we would like to confirm in vivo the PCA of human liver MSCs in a rat model, then study it’s inhibition by a specific anticoagulant cocktail and finally assess the effect of this cocktail on cell implantation. The main aim of this study is to control the PCA induced by infused liver MSCs without reducing the implantation of cells. Methods: Wistar rat were transplanted with 50x106/kg fluorescent (cell tracker red) human liver MSCs by intraportal infusion with (n=12) or without (n=13) anticoagulant drugs. Using an intra-femoral catheter we injected FITC-dextran and Hoechst to visualise liver vasculature and cell nuclei. By intravital microscopy (IVM) we analysed the left liver lobe at different time points, 1h-24h-48h and 7days post-infusion to study liver MSCs localisation and their effect on liver microvasculature. By pathological examination, cell localisation, cell implantation, vascular and tissue alterations were studied at the same time points. Serial slides were performed for a standard haematoxylin eosin staining and a human cell staining (human B-1-integrin). Results: By IVM we observed that the transplanted cells in the first hour formed aggregates in the larger liver vessels, then cells migrate to the sinusoids after 24h, to be quickly cleared after 7days. After 24h, large defect of perfusion were observed in both groups, but normal hepatic vascularisation was restored after 48h. These observations were confirmed by pathological examination. Large necrotic zones surrounding the infused cells were observed after 24h, with respectively 14.7% and 19.5% of the liver tissue in the non anticoagulated and anticoagulated group. This necrosis decreased rapidly after 48h to 0.5% and 1.9%. The anticoagulant drugs didn’t prevent this necrosis, no difference has been observed between the 2 groups (p>0.05).We confirmed that the infused cells are rapidly cleared after 24h from the liver over time, with respectively after 1h, 24h, 48h and 7days a decrease from 1.3%, 0.3%, 0.07% till 0.03% of infused cells. No difference in cell implantation has been observed between the groups with or without anticoagulation (p>0.05). Conclusion: Intraportal infusion of human liver MSCs to Wistar rats induces a transient alteration in liver vascular flow after 24h. This could be explained by the temporary localisation of liver MSCs in large portal veins and sinusoids up to 1hour after the infusion, in addition to a possible xenotransplantation acute reaction. After 24h more than 70% of cells are cleared and cells are surrounded by transient necrotico-hemorragic regions that regress almost completely after 48h. After 7days no necrosis and very few cells are seen. We observed no difference between the two groups, with or without anticoagulation.