Hypoxia Preconditioned Urine-derived Stem Cells Accelerate The Recovery Of Chronic Liver Injury In Mice

To compare the biological characteristics of urine-derived stem cells(USCs)under normoxia and hypoxia culture conditions in vitro,and to explore whether hypoxia precondition can promote the recovery effect of USCs transplantation in the treatment of mouse chronic liver injury model and its possible mechanism.Methods: 1.Urine samples were collected from 10 healthy men aged20~25,USCs were separated and cultured.Cell morphology was observed under microscopy.Flow cytology was used to detect the stem cell surface markers.Alkaline phosphatase(ALP)staining,Alizarin red staining,oil red O staining,ICG uptake release test and PAS staining were performed to evaluate osteogenic,adipogenic,and hepatic differentiation of induced USCs.Laser confocal microscope was used to detect cell-cell fusion of USCs and different liver cells in vitro.The nude mice model of chronic liver injury was established by intraperitoneal injection of CCL4,twelve weeks after CCL4 injection,nude mice in the transplantation group were injected with USCs via the tail vein twice a week.Liver tissues were takenat 2 weeks after first cell injection,liver index was calculated.Serum samples were collected for measurements of ALT and AST.HE and Masson staining were performed to determine the pathological changes.Frozen sections were used to observe cell-cell fusion of USCs and hepatocytes in vivo.2.To compare the cell morphology and biological characteristics of USCs in normoxia and hypoxia culture conditions respectively.Chronic liver injury nude mouse model was successfully established.The normoxia and hypoxia pretreated USCs were injected via the tail vein twice a week.The serum and tissue samples were obtained at two weeks after first cell injection,and the liver index was calculated.Serum AST and ALT were detected,histological detection was performed to determine the liver pathological changes,fibrosis and oxidative stress related proteins were detected by immunohistochemistry.3.Autophages and autophagic lysosomes were observed by transmission electron microscopy(TEM).Western Blot(WB)was used to detect the expression of autophagy-related proteins LC3 and Beclin1.After cells were transfected with ptfLC3(dual-fluorescene mRFP-GFP-LC3plasmid),the changes of autophagy were observed by confocal microscopy.Autophagy inhibitors 3-Methyladenine(3-MA)and bafilomycin(Baf)pretreated cells for 3 hours,and then cells were cultured under hypoxia condition for 48 hours.Cell proliferation ability was measured by trypanblue staining and clone formation assay.Cell apoptosis and cell cycle were detected by flow cytometry.Wound-healing and transwell migration assay were used to detect horizontal and vertical migration ability.The expression of autophagy-related proteins LC3,Beclin1 and P62 was detected by WB,and the osteogenic and adipogenic differentiation potentials were assessed by ALP staining,alizarin red staining,and oil red O staining.Results: 1.USCs derived from normal healthy men were rice-like adherent growth cells,expressing mesenchymal stem cell surface markers.With induction of osteogenesis and adipogenesis,part of USCs were positive in ALP staining,alizarin red staining and oil red O staining.USCs co-cultured with hepatic stem cells had positive ICG and PAS stains,suggesting that USCs have osteogenic,adipogenic and hepatic differentiation potential.Cell-cell fusion existed in mixed co-culture of USCs and different hepatocytes in vitro.In the chronic liver injury nude mouse model,the liver index of the model group was significantly increased(p<0.05)compared with the control group,the acute liver injury marker ALT statisticlly increased(p>0.05),and the chronic liver injury marker AST slightly increased(p<0.05).The hepatocyte degeneration appeared as disordered hepatic cord structure,nuclear pyknosis or disappearance,more island-like pseudolobular structures with infiltration of inflammatory cells in portal area.Masson staining showed plenty of bluestained fibrosis around the portal area.With USCs transplantation,liver index decreased(p <0.05),the value of ALT and AST decreased with no statistic difference(p> 0.05).The degeneration of hepatocytes and the fibrosis in the transplantation group obviously recoveried compared with that in the model group.Immunofluorescence revealed that exogenous USCs in the liver of the transplantation group positively expressed albumin(albumin,ALB),and PKH26 labeled USCs were detected as GFP positive cells in green fluorescent protein transgenic mice,indicating that USCs and hepatocytes have cell-cell fusion phenomenon in vivo.2.The cell morphology of USCs continuously cultured in hypoxia condition were smaller and more homogeneous compared with that in normoxia condition.Flow cytometry showed no significant difference in the expression of CD markers in USCs under two culture conditions.Cell growth curves in two groups showed logarithmic growth trend,cells in hypoxia group had stronger cell proliferation and migration ability.After 48 hours of hypoxia preconditioning,the percentage of cells in S phase increased significantly,and the apoptotic rate had no significant change.With osteogenic,adipogenic and chondrogenic induction,ALP,alizarin red,oil red O and alcian blue staining of the two groups were positive and had no significant difference.In the chronic liver injury model,the number of cells colonized in liver tissues of the hypoxia pretreated group was more than that of the normoxia group.With USCs transplantation,the liver indexand the level of ALT had no significant change,serum AST level was partly recovered,but there was no significant difference between the two transplantation groups.HE and Masson staining showed that the pathological structure disorder and blue stained collagen fibers of the two transplantation groups significantly recovered compared with the model group,the blue stains were less in hypoxia group than that in normoxia group.The basic expression level of fibrosis marker ?-smooth muscle actin(?-SMA),oxidative stress related genes MPO and 8-OHdG was very low in normal liver,which obviously increased in the model group,after USCs transplantation,the expression of ?-SMA?MPO and 8-OHdG significantly reduced,but there was no significant difference between the two USCs transplantation groups.3.After 48 h of hypoxia pretreatment,a large number of "autophages" with bilayer or multi-layer membrane structure and autophagic lysosomes could be observed under TEM.The autophagy maker protein levels of Beclin1 and LC3-II significantly increased.USCs were transfected with ptfLC3 double fluorescent plasmid,confocal laser scanning showed that more autophages with yellow spots and autophagic lysosomes with red spots could be seen in the hypoxia group than the normoxia group.The above results suggested that hypoxia treatment could significantly enhance the autophagy level of USCs.After inhibiting autophagy with 3-MA and Baf,the expression of Beclin1,P62 and LC3 significantly decreased in the3-MA and Baf groups,cell proliferation was significantly decreased,the number of cell clones decreased,and the average diameter of clones significantly decreased,especially in the Baf group.The wound healing time of 3-MA and Baf groups was longer than that of the normoxia group,and the number of transwell migration cells significantly decreased compared with the hypoxia group.There was no significant difference in apoptosis and cell cycle between the four groups.Hypoxic precondition upregrulated cell autophagy would not affect the differentiation potential of USCs.Conclusion: This study has confirmed that urine-derived stem cells have similar cell phenotype of mesenchymal stem cell,and the characteristic of self-renewal and multi-directional differentiation potential.USCs transplanted into chronic liver injury model mice can effectively improve liver function and repair liver tissue injury to a certain extent.Hypoxia pretreatment might promote cell proliferation and immigration by inducing cell autophagy,thereby improving the colonization survival rate of USCs in injured liver,and then improving the repair effect of USCs transplantation therapy.Autologous urine-derived stem cells obtained by non-invasive method may have the potential to be used as an ideal seed cell source for hepatocellular transplantation,showing a good application prospective in the clinical treatment of chronic liver diseases.