Research On Hepatectomized Patient Sera In Promoting Hepatocyte Proliferation And Differentiation From HiPSCs

Orthotopic liver transplantation is the most effective treatment for liver failure which is limited by the shortage of donors. Hepatocyte transplantation and bioartificial liver (BAL) as auxiliary treatments for liver transplantation, both need functional hepatocytes. But primary hepatocytes are difficult to proliferation and lose function immediately in vitro. Embryonic stem cells (ESCs) can be differentiated into liver cells whose application is limited by ethics and immune rejection defects. IPSCs have almost all characteristics of ESCs and supeoior to ESCs for avoiding the ethical issue and may avoid immune rejection. Though iPSCs can be successfully induced into hepatocytes, the induction efficiency and function of HLCs is very low.Objective:1. Establish stable culture system for hiPSCs and stable method for differentiating hiPSCs to HLCs.2. Find better method of inducing human iPSCs into HLCs to improve the induction efficiency and HLCs’functionality.3. Find better culture conditions to maintain functionality and proliferation ability of liver cells.Methods:1. In feeder culture system, embryonic fibroblast derived from CF-1mouse was used as feeder cells, fibroblast growth factor (FGF) and beta mercaptoethanol was added in the DMEM/F12medium. In feeder-free culture system, hiPSCs was cultured in mTeSRl medium combined with Matrigel matrix.2. Mimic the embryonic development of mammalian liver, the induction process was divided into three stages including definitive endoderm, hepatoblast and mature hepatocyte, sequentially supplemented with activin A, bFGF, hepatocyte growth factor (HGF) and dexamethasone to the culture medium.3. Serum from patients undergoing hepatectomy (acquired pre-hepatectomy,3hours,1day,3days post hepatectomy) was used to replace fetal bovine serum (FBS) when differentiating hiPSCs into HLCs. Properties of HLCs were assessed and compared with cells cultured in FBS.4. Serum from patients undergoing hepatectomy (acquired pre-hepatectomy,0.5hours,3hours.24hours,72hours post hepatectomy) was used to replace FBS in the medium culturing HL-7702cells. Proliferation of HL-7702cells cultured in different serum was compared through cell sorting and BrdU testing.Results:1. In feeder and feeder-free culture systems, alkaline phosphatase staining and Oct4immunofluorescence staining of the hiPSCs were both positive.2. We successfully established a stable method of differentiating human iPSCs into HLCs. The induction efficiency was18.1%. Cells induced from hiPSCs can express ASGPR1, albumin and AFP. also have glycogen synthesis activity and ICG uptake function.3. Flow cytometry analysis show that except for POST3D-HS group, ASGPR1positivity among hiPSC-derived HLCs induced by human serum was higher than that induced by FBS. Cells cultured with human serum showed significantly higher ALB secretion compared to the FBS group (P<0.05), and that in POST3H-HS and POSTID-HS groups was significantly greater than that in PRE-HS group (P<0.05). Urea synthesis function in POST3H-HS and POSTID-HS groups were higher than that in FBS group and PRE-HS group (P<0.05). ALB expression intensity in human serum groups was greater than that seen in FBS group, and the proportion of ALB-positive cells in POST3H-HS and POSTID-HS groups were significantly greater than that in FBS and PRE-HS group. AFP expression intensity in POST3H-HS and POSTID-HS groups was greater than that seen in FBS group and PRE-HS group. POST3H-HS and POSTID-HS groups showed enhanced glycogen synthetic activity and increased ICG uptake function compared with the FBS group and PRE-HS group.4. Living cell image analysis and BrdU immunofluorescence staining both showed that multiplication rate of HL-7702cells cultured in human serum was superior to those cultured in FBS (P<0.05). Those cultured in human serum0.5hours and3hours post hepatectomy were superior to those cultured in human serum pre-hepatectomy(P<0.05).Conclusion:1. Human serum, particularly that acquired relatively soon after hepatectomy (3hours and24hours post hepatectomy). can enhance differentiation efficiency and functionality of HLCs derived from human iPSCs.2. Human serum can promote the proliferation of hepatocytes compared to FBS. The influence of serum acquired post hepatectomy closely associated with the postoperative time.