In Vitro Hepatic Differentiation Of Human Bone Mesenchymal Stem Cells With Oxalate-degredation Genes

Background: Primary hyperoxalurias (PHs) is a rare aytosomal recessive inheritedcondition due to a functional deficiency of certain liver-specific enzyme, such asalanine glyoxoylate aminotransferase (AGT). The disease is characterized byoverproduction and accumulation of oxalate in the body that results in recurrenturolithiasis and/or progressive nephrocalcinosis and/or renal failure. With themetabolic defect in the liver and the problem being overproduction of oxalate withoutother avenues for its disposition, it is clearly necessary to perform liver or combinedliver/kidney transplantation. However, liver transplantation has several limitationssuch as difficulties of the operation, high surgical risk and several majorcomplications. Cell transplantation is less invasive than whole organ transplantation.Liver cell transplantation can be developed into a procedure that could be used forroutine bridging to orthotopic liver transplantation or, even better, as an eventual curefor primary hyperoxaluria type I. Unfortunately, limitation of cell sources, short-termviability and rapid phenotypic dedifferentiation of hepatocyte are the major obstaclesfor clinical application. The human mesenchymal stem cells (hMSCs) candifferentiatie into wider variety of cell types under some experiment conditions. Thereis increasing evidence to suggest that hMSCs may be transplantable source of hepaticprogenitor cells. Oxalobacter formigenes (Ox.F) are microorganisms that have apowerful oxalate-degrading capability. Ox.F’s oxalate-degrading genes are oxc andfrc, which, respectively, express as oxalyl-coenzyme A decarboxylase (OCoAD) andformyl-coenzyme A transferase (FCoAT). In our previous research, the oxalate- degrading genes of Ox.F-the frc gene and oxc gene–were transfected into hMSCsto give the latter an oxalate-degrading function. In this research, we would do furtherwork to comfirm that hMSCs-frc-oxc could be differentiated into hypatocyte in vitroand retained the function to degrade oxalate.Objective: To induce the differentiation of hMSCs with oxalate-degrading genes fromOx.F (hMSCs-frc-oxc) into hepatocyte-like cells in vitro and then investigate theoxalate-degrading effects of the treated cells in the hope that it could be used to treathyperoxaluria, expecially PHs.Methods: The hMSCs-frc-oxc in passage4were cultured and induced to differentiateinto hepatocyte-like cells in differentiation medium supplemented with40ug/Lhepatocyte growth factor (HGF) and10ug/L fibroblast growth factors-4(FGF-4). Themorphological changes of cells were observed microscopically at different periods.The expression of hepatic markers [alpha fetal protein (AFP), albumin(ALB) andcytokeratin-18(CK-18)] and oxalate-degrading genes (frc and oxc) were separateddetected by immunocytochemistry and Western blotting at different times afterinduction, and the expression of AFP, ALB, CK-18, frc and oxc mRNA by real-timequantitative polymerase chain reaction (RT-PCR). Finally, the oxalate-degradingfunction of treated cells was analyzed by ion chromatography. The human liver L-02cells and transferred mock-vehicle hMSCs (hMSCs-vector) were used as positive andnegative controls, respectively.Results: The hMSCs-frc-oxc were cultured successfully in vitro. hMSCs-frc-oxcgradually transformed into polygonal cells with the process of induction. After hepaticdifferentiation, the hMSCs-frc-oxc expressed not only hepatocyte-specific marker(AFP, ALB, CK-18) but oxalate-degrading genes (frc and oxc) at both mRNA andprotein levels as well. Furthermore, the treated cells had kept the function of oxalatedegradation which was tested by ion chromatography. The human liver L-02cells andhMSCs-vector, by contrast, had little function to degrade oxalate.Conclusion: The hMSCs with oxalate-degrading genes from Ox.F could be differentiated into partially functional hepatocyte-like cells in vitro successfully.Meanwhile, the hepatic differentiated hMSCs-frc-oxc can not only express the oxcgene and frc gene at both mRNA and protein levels but also have retained the abilityto degrade oxalate. The successful hepatic differentiation of hMSCs-frc-oxc in vitromight be used in the etilogical therapy of PHs and provides wide cell sources for cellimplantation in treatment of PHs.