Mechanistic Study Of The Acute And Chronic Liver Injury On The Biodisposition Of Mesenchymal Stem Cells

Cell-based therapies were expected as a“third pillar”of medicine in the future.Among them,Mesenchymal Stem Cells were especially attractive owing to their abundant source,multi-lineage differentiation potential,low immunogenicity and immuno-modulatory characteristics^[1,2].However,Emerging evidence demonstrated the substantial nonresponse rate and obvious individual variance in MSC-based clinical trials^[3].Pharmacokinetics（PK）study which focuses on details in the dynamic movements of the drug will also provide valuable information for cell therapy in efficacy improvement,mechanism exploration and safety evaluation.The investigation in the distribution of MSCs in vivo will help us explaining the unreliability and instability in clinical to some extent.Meanwhile,there are some indications in previous studies regarding the disposition and elimination mechanism of MSC but most of them lack strong experimental evidence.In this study,an optimized quantitative method was employed to examine the disposition of intravenous injected MSC in vivo.The impact of various diseases on the pharmacokinetics of exogenous MSC and potential mechanisms were further explored.Firstly,qualitative and quantitative methods with the combination of Cy5-NHS labeling and q PCR were established to detect the exposure of transplanted cells in the tissues.By comparing with traditional Di R dye and considering the influence of matrix effect on the accuracy of q PCR,our refined method was proved to be highly specific and sensitive,stable and repeatable.Then the in vivo distribution of administered h MSC（5×10⁵ cells/mice）was elucidated in C57/BL6 healthy mice.The result showed that most of the injected cells were accumulated in the lung（around 90%）and liver（around3%）while a negligible amount was detected in other organs（<0.5%）.A significant double peak of MSC emerged in the lung within 1-2 hours after intravenous injection.Only a small fraction of h MSC（less than 5%）was found to survive in the body.The short half-life,quick elimination and low bioavailability（BA）might contribute to the poor clinical performance of MSC.Furthermore,whether the PK behaviors of MSC would be altered by liver diseases was investigated as well.The acute liver failure and chronic fibrosis model were induced in Balb/c mice by a single or repeated administration of carbon tetrachloride（CCL₄）separately.It could be found that in CCL₄-induced fulminant hepatitis mice,the engraftment of MSC in the lung was significantly improved with a 50%and 40%increase in C_max and AUC_0-last,and the figures in the liver were 7.9%and 163%respectively.The serum ALT and AST level and the m RNA expression of inflammation-related factors were significant reduced after h MSC administration which profiled its immunomodulatory effects in liver inflammation.However,in the chronic injury experiment,the exposure in the lung from the fibrosis+h MSC group mice was dramatically lowered.The C_max and AUC_0-last,of h MSC were decreased by approximately 32.2%and 60%.Thus,the protective activities of a single dose of h MSC were limited in the liver and lung where the inflammatory infiltration and fibrosis occurred.It might be easier for cells to be removed from the body in fibrosis mice.The hypothesis of whether the difference in exposure diversity and efficacy were associated with the MSC clearance alteration was further explored.Natural killer cells are a vital subset of immune cells which was reported to mediate the lysis of MSC in cell experiment before.Therefore,we investigated whether the difference in PK was attributed to the NK cell-mediated clearance.Flow cytometry and immunofluorescence analysis showed significant inhibition of NK cell in liver failure mice while the opposite was the case in the chronic injury compared with the control group.Interestingly,similarities were that an improved cell amount with NK downregulation in acute injury mice for intravenous m MSCs.To confirm the essential role of NK cells in the MSC elimination,an anti-ASGM-1 antibody was utilized to deplete the activities of NK cells and brought more h MSC in lung and liver compared with control.Finally,the connection of the NK cell-mediated MSC lysis with the N-linked glycosylation of target MSC was built in vitro and in vivo,which provides new ideas for developing strategies to enhance the residence and protective effects of stem cells.In conclusion,the study brings new interpretations for the unstable efficacy and significant individual difference in terms of MSC-related treatment in various diseases.And we for the first time evidenced the NK cell-mediated destruction of MSC in vivo and in vitro and demonstrated the clearance mechanism for MSC.This will expand the current understanding of MSC-based therapy and provide a theoretical foundation for the optimization of liver disease.