Study On The Targeted Differentiation Of Hipscs Mediated By Nanoparticles To Hepatocytes

Since Yamanaka first used the "four factor" combination(SOX2,OCT4,C-MYC,KLF4)to reprogram somatic cells into induced pluripotent stem cells(i PSCs)in2006,cell reprogramming has been a hot topic in the field of life science and regenerative medicine.Since iPSCs are derived from the patient’s own somatic cells,it has the ability of self-renewal and multi-directional differentiation.Therefore,the application of iPSCs as an alternative to human embryonic stem cells(hESCs)in the clinical application of seed cells,to avoid the ethical and legal issues involved in hESCs,and iPSCs are derived from patients themselves,to achieve personalized treatment.Orthotopic liver transplantation is the only effective method for the treatment of liver cancer.However,due to the shortage of organs,the number of deaths caused by liver cancer has been increase in recent years;In addition,the incidence of liver cancer is rising in the world.Clinical trials of hepatocytes transplantation have brought encouraging results.However,hepatocytes transplantation as an alternative to liver transplantation still needs to address the problem of hepatocytes origin.At present,many researches have been devoted to finding a feasible method to produce stable functional hepatocytes.One of the cells that may be an inexhaustible source of hepatocytes are human induced pluripotent stem cells(hi PSCs).hi PSCs has the ability of self-renewal and multi-directional differentiation.Compared with human embryonic stem cells(hESCs),hiPSCs involves less ethical problems and is completely independent of the source.Therefore,hepatocytes derived from hiPSCs(hiPSC-HEPs)have important significance in drug screening,cell therapy and disease models in vitro.This research is based on calcium phosphate nanoparticles as gene carrier,carrying two different factor combinations to reprogram human umbilical cord mesenchymal stem cells(hUMSCs)into hi PSCs,then the hiPSCs were induced into hepatocytes,and the best differentiation medium was optimized.The best induction scheme was used to culture the cells,so as to provide a theoretical basis for liver transplantation.Chapter 1 Review on preparation of human hepatocytes by reprogramming method and its applicationBased on a large amount of data,this paper reviews the methods of reprogramming human hepatocytes,and summarizes and analyzes the influencing factors of human hepatocytes reprogramming.This paper reviewed the research status of human hepatocytes reprogramming technology and its application in regenerative medicine,drug screening and disease model in vitro,which provide a theoretical basis for the smooth development of the experimental work.Chapter 2 Reprogramming of hiPSCs based on non-viral nanopartical gene delivery systemUsing calcium phosphate nanoparticles as gene carrier,comparing two different factor combinations,namely pSOCK(SOX2,OCT4,C-MYC,KLF4)and pOS+miR(OCT4,SOX2,miR302b-367),C-pSOCK and C-pOS+miR nanoparticles were prepared and characterized by transmission electron microscopy,agarose gel electrophoresis,toxicity evaluation,particle size and Zeta potential measurement;Human umbilical cord mesenchymal stem cells(hUMSCs)were used as the source cells for reprogramming.The results showed that the nanoparticles prepared by the combination of the two factors showed a regular,uniform,spherical or ellipsoidal surface with positive charge;Cytotoxicity test showed that the calcium phosphate nanoparticles prepared by two different factor combination were non-toxic or low toxicity;The number of positive clones was counted by alkaline phosphatase staining,so as to investigate the reprogramming efficiency of the two different nanoparticles;Immunofluorescence and Western-blot showed that hiPSCs induced by C-pOS+miR nanoparticles could express pluripotent markers of embryonic stem cell,and could differentiate into three germ layers in vivo(inner and outer,differentiation).Chapter 3 Study on hiPSCs differentiation into hepatocyteFour different differentiation mediums were set up(Medium I,II,III,IV)to differentiate hiPSCs into hepatocytes.By using enzyme linked immunosorbentassay(ELISA)respectively in the differentiation of day0,3,7,11,15,19,23,27,32 to detect AFP and ALB expression,which selected the best medium and the best induction scheme;The morphological changes of cells were observed under inverted microscope;The expression of liver specific proteins(AFP,ALB,CK8,CK18,SOX17)were detected by immunofluorescence and Western-blot.The results showed that the induction Medium II was better;The morphological changes of cells were observed under microscope,the morphology of cells from the "clone" shape gradually changed to "paving stone" shape,after induced differentiation,cell morphology is basically the same as the multi angle polygon or round,and the nucleus is obvious,some cells showed typical liver cell morphology: dual core or multi-core phenomenon,and the differentiation of liver cells can express liver specific protein(AFP,ALB,factor CK8,CK18,SOX17).Chapter 4 Study on the expression and activity of drug metabolizing enzymes in human hepatocytes in two dimensional culture systemCYP450,UGT and GST were selected under the two-dimensional culture system,and the expression of protein were determined by Western-blot,ES-HEPs were used as positive control;The dynamic changes of drug metabolizing enzymes were observed at different time during differentiation,comparing the difference of the expression of drug metabolizing enzymes between hiPSCs-HEPs and hESCs-HEPs;The expression of AST,ALT and LDH were measured at different time points;The contents of albumin secretion,urea synthesis and glucose comsumption were measured by using the method of bromo cresol green,two acetyl oxime and glucose oxidase peroxidase.The results showed that hepatocytes derived from hiPSCs(hiPSCs-HEPs)could express CYP450,UGT and GST,and there was no significant difference between the expression of hESCs-HEPs;However,hiPSCs before induction did not express or even express CYP450,UGT and GST;The changes of AST,ALT and LDH during the differentiation of hiPSCs were consistent with that of hESCs;The levels of albumin secretion,urea synthesis and glucose consumption were similar to those of hiPSCs-HEPs and hESCs-HEPs.Chapter 5 Study on the function and activity of hepatocytes in three dimensional culture systemFor the first time,the hiPSCs generated by reprogramming were induced to differentiate into hepatocytes in the three-dimensional culture system.The collagen-chitosan three-dimensional scaffolds were prepared by freeze-drying and thermosetting using collagen Ⅰ and chitosan as material.The effect of different quality ratio and different curing time on the scaffold performance was studied.The three-dimensional porous scaffold preparation process was carried out.The nanoparticles C-pOS + miR were adsorbed on the inner surface of the blank collagen /chitosan scaffolds,and the three-dimensional carrier nanoparticles-collagen /chitosan was prepared by observing the morphology of the scaffolds.(HUMSCs)were used to reprogram in the three-dimensional gene-loaded nanoparticles-collagen /chitosan scaffolds to form clear and regular clones.Induced directly hiPSCs differentiation into hepatocytes in the three dimensional scaffolds,comparing hepatocytes function between the two and three dimensional culture system by ELISA;The metabolic activity of hepatocytes in two and three dimensional culture systems was studied by using the method of bromo cresal green,two acetyl oxime and glucose oxidase peroxidase respectively.The results showed that after differentiation 21 days in three-dimensional culture system,cell morphology showed hepatocytes-like cell morphology significantly;In three-dimensional culture system,the function of hepatocytes differentiated from hiPSCs were "no damage",and still in the normal liver function.