Establishment Of Induced Pluripotent Cells (iPSCs) And Ipscs-derived Hepatocytes Differentiation And Transplantation Immunity Study

PART Ⅰ Isolation,culture and identification of primary human hepatocytesOBJECTIVE:To optimize the method of separating hepatocytes on the basis of currently feasible tissue perfusion methods to obtain higher yield and activity of hepatocytes.To identify whether the isolated primary hepatocytes can maintain the specific phenotype of hepatocytes in vitro and to exert some function of hepatocytes.Methods:By studying the existing methods of human hepatocytes isolation from tissue,and adjusting the enzyme composition and concentration,the perfusion route and time,the centrifugal conditions and so on,the isolation of the primary hepatocytes from the liver tissue yield and cell activity were compared and analyzed,and deduced the efficient method of hepatocytes isolation from tissue.The isolated primary hepatocytes were cultured in vitro and continuous observation of its growth state.The expression of ALB,AFP and ASGPR1 protein in hepatocytes were detected by immunofluorescence staining.And the changes of CYP450 activity before and after the drug were compared and analyzed,to evaluate whether the human primary hepatocytes could culture in vitro and have good hepatocyte phenotype and function.Results:The perfusion method of primary hepatocytes isolation from human liver were improved and optimized.The cell yield was(20-50)×106/g wet weight,and the cell viability was more than 90%.Through the continuous culture and observation in vitro,it was shown that the isolated and cultured primary cells had the specific morphology of the hepatocytes and could be maintained for about 4-7 days.The high expression of ALB and ASGPR1 on the surface of isolated hepatocytes were detected by immunofluorescence staining,and low expression of AFP was confirmed.The results showed that the isolated and cultured hepatocytes by the modified method were similar with the phenotype of hepatocytes.In addition,the induction of CYP450 in cultured human hepatocytes in vitro showed that the obtained human hepatocytes could exert a high pharmacological activity in vitro,and further demonstrated that the isolation method could obtain higher quality hepatocyte.Conclusion:High-quality hepatocytes were successfully isolated from human liver tissue by adjusting the existing hepatocytes separation methods from tissue.Primary hepatocytes can be cultured in vitro for a certain period of time to maintain similar cell phenotype of hepatocytes and to exert hepatocyte-related drug metabolism.PART Ⅱ Establishment and identification of induced pluripotent cells(iPSCs)from human skin fibroblastOBJECTIVE:To investigate the possibility and efficiency using noncompliant transfection technique with additional vectors of OCT3/4,SOX2,KLF4,L-MYC,LIN28 and p53 short hairpin RNA(shRNA)in reprogramming human skin fibroblasts to induced pluripotent stem cells(iPSCs).METHODS:Fibroblasts were isolated from human skin tissue by enzymatic digestion in vitro and fibroblasts were identified by detecting Vimentin protein expression.The plasmids containing eGFP,OCT3/4,SOX2,KLF4,L-MYC,LIN28 and p53 shRNA were purified and transfected into human skin fibroblasts by nuclear transfection.The expression level of eGFP was observed under fluorescence microscopy to evaluate the transfection efficiency.Under the condition of iPSCs culture,the growth state of the reprogrammed cells was observed and changed everyday.When the iPSCs colony formation and appeared,the micro-selection was carried out and the cells were continued to culture.The alkaline phosphatase staining was detected in the established iPSCs.The specific protein of pluripotent stem cells NANOG,OCT3/4,SOX2,SSEA-4 and TRA-1-60 in the established iPSCs were identified by immunofluorescence staining and the expression of OCT3/4 and NANOG gene were measured by RT-PCR.In addition,the pluripotency of the established human iPSCs was determined by the teratoma formation experiment.Results:The cells isolated from human skin tissue and cultured in vitro were shown that the morphology of fibroblasts and express the Vimentin protein.The recombinant plasmid was successfully transferred into human fibroblasts by nuclear transfection.The expression level of eGFP was stable at 20%-30%at the second day after transfection.Through continuous observation,the iPSCs colony formation was found under light microscope on the 28th day after reprogramming,and the amplified cells showed the growth status of iPSCs.Alkaline phosphatase staining revealed that reprogrammed cells showed blue positive expression.The results of immunofluorescence and RT-PCR showed that the high expression of NANOG,OCT3/4,SOX2,SSEA-4 and TRA1-60 protein and OCT3/4 and NANOG pluripotency genes.In addition,the reprogrammed human iPSCs were injected subcutaneously and then formed tumor-like tissues in mice.Histological analysis showed that the three embryonic-derived derived tissues could be found in the tumor.CONCLUSION:Human fibroblasts can be isolated and expanded from the skin tissue in vitro.Additional plasmids carrying OCT3/4,SOX2,KLF4,L-MYC,LIN28 and p53 shRNA can be successfully transferred into human skin-derived fibroblasts by nuclear transfection and efficiently reprogrammed like iPS cells.In addition,the established iPSCs showed iPSCs specific cell phenotype in a series of experiments in vitro,and in vivo experiments showed that reprogrammed cells were pluripotent.PART Ⅲ Differentiation and transplantation of human induced pluripotent stem cell(iPSCs)-derived hepatocytesOBJECTIVE:To investigate a feasibility method with more simple,time-saving and high efficient of differentiation from human-induced pluripotent stem cells(iPSCs)into hepatocytes,and to analyze the possible factors influencing the differentiation efficiency in this process.In addition,the differences between iPSCs-derived hepatocytes and tissue primary hepatocytes were further studied to guide the research of hepatocyte differentiation from iPSCs and to identify the cytological characteristics and application of iPSCs derived hepatocytes.Methods:Reference to the existing hepatocytes differentiation program from iPSCs,the program of obtaining high-quality iPSCs derived hepatocytes was summarized by adjusting and combining the initiation state of differentiation conditions,induced cytokine application and concentration,induction time of the various stages,and screened specific markers at the end of each stage.The expression levels of stem cell pluripotency genes and hepatocyte specific genes in iPSCs-derived hepatocytes were identified by RT-PCR.The expression of hepatocyte specific proteins ALB,ASGPR1 and AFP in iPSCs-derived hepatocytes were detected by immunofluorescence staining,and the secretion ability of ALB protein in iPSCs-derived hepatocytes was quantitatively analyzed by ELISA.The metabolic activity of CYP450 in iPSCs-derived hepatocytes was then observed by drug induction in vitro.In addition,the expression of human ALB protein in transplanted mice was observed in the mice with partial hepatectomy after cell transplantation.It was evaluated whether iPSCs derived hepatocytes could homogenize and proliferate in the liver of mice and performant hepatocytes function.Results:Compared the condition of the hepatocytes differentiation from iPSCs,it was found that the cells were able to stably express more than 90%of endoderm-specific protein SOX17 after the first stage when the pre-differentiated cell confluence was about 20%.There was no significant difference between 20 ng/mL and higher concentration of HGF in the second stage.At the end of second stage,the differentiated cells began to show morphological changes of hepatocytes and stable expression of about 80%HNF4 a protein.The low expression of OCT3/4 and NANOG were confirmed by RT-PCR,and the high expression of hepatocyte-specific genes such as ASGPR1,TAT,CYP3A4 and CYP7A1 were observed.It was found that the expression of ALB,ASGPR1 and AFP protein in the iPSCs-derived hepatocytes showed the derived hepatocytes were close to the mature state of hepatocytes,and that the derived hepatocytes also had the secretion function of ALB protein,the expression level is close to 70%of the activity of isolated primary human hepatocytes.In addition,in the mice of liver injury with retrosine pretreated,iPSCs derived hepatocytes can homogenize and proliferate in liver after spleen injection,and also can secrete human ALB protein and gradually increase with time.The results indicated that iPSCs derived hepatocytes can play a certain role ofhepatocyte function in the vivo environment.CONCLUSION:Under artificial conditions,iPSCs can efficiently differentiate into hepatocytes through improved protocols,and the protocolare optimized in time,equipment and reagent applications.In addition,the phenotype and hepatocytes function of iPSCs derived hepatocytes in vivo and in vitro are similar to primary hepatocytes,but its induction efficiency and biosafety need further study.PART Ⅳ Effects of regulation of BATF expression on acute rejection in allograftsOBJECTIVE:To investigate the effect of BATF gene on acute rejection in allografts and its possible mechanism.Methods:The model of acute rejection was constructed by transplanting the heart of BALB/c(H2d)mice into the peritoneal cavity of C57BL/6(B6,H2b)mice on the basis of mouse model of heterotopic heart transplantation.The BATF shRNA vector was constructed in vitro and its interference efficiency was identificated.Mice were divided into three groups:BATF shRNA,Negative shRNA and Control group,and the mice were treated with corresponding treatments.The survival of the transplanted heart was assessed by daily touch of heart beat.The occurrence and extent of acute rejection were examined by histological examination.The expression of Th1,Th17 and Treg cells in the spleen of each group was detected by flow cytometry.The expression of transcription factors and secretion of cytokines were analyzed by RT-PCR and ELISA respectively.Results:The animal model of allograft rejection was successfully constructed on the basis of mouse heterotopic heart transplantation.The three kinds of BATF shRNA vectors were successfully constructed and the shBATF vector with high efficiency was screened out.The study found that the allograft heart can be extended under the action of shBATF for about 3-5 days,HE staining analysis of cardiac tissue showed that the inflammatory cells infiltration significantly reduced in BATF shRNA group mice after transplantation.In addition,the expression of Thl cells in the splenic lymphocytes of mice was not found significantly different by flow cytometry,while the expression ratios of Th17 and Treg cells were decreased and increased respectively.There was a statistically significant difference.The results of RT-PCR and ELISA also showed that the corresponding transcription factors and cytokines in these cells also showed similar expression.In addition,the secretion of cytokine IL-4 in serum of BATF shRNA treated mice was significantly decreased.Conclusion:The inhibition of BATF expression can prolong the survival of allografts and decrease the degree of acute graft rejection.The mechanism may be related to the changes of Th17 and Treg cell axis and the decrease of IL-4 secretion.