Liver Extracellular Matrix Prompts Pluripotent Stem Cells Differentiation Into Hepatocyte-like Cells

The liver is the largest "chemical factory" in the body,carrying four important functions,including synthesis,metabolism,detoxification,and secretion.Liver disease,characterized by extensive disease spectrum as well as complicated condition,is hazardous to human health.End-stage liver diseases lead to decompensation of liver function,liver fibrosis,and even induce malignant tumors.So far,liver transplantation is still the only effective treatment for end-stage liver disease.However,liver transplantation is limited by a series of issues,such as the serious shortage of donor liver sources,the impact of liver transplantation trauma,immune rejection after transplantation and so on.In recent years,hepatocyte transplantation and bioartificial liver replacement therapy have been developed and can be used as an auxiliary method for liver transplantation,partially solving the above problems.However,the source of human-derived hepatocytes is also very tight and they are difficult to isolate.Moreover,the primary hepatocytes have only a part of the proliferation activity,making it difficult to achieve large-scale production and application.Additionally,the function of hepatocytes is difficult to maintain in vitro,and de-differentiation will occur over time.Therefore,it is very important to find a suitable source of liver cells.The ideal candidate cells should be easy to expand in vitro,and the differentiated cells should have adequate liver function and weak immunogenicity.Also,they should be convenient for long-term storage or transportation,and can participate in liver tissue reconstruction after transplantation.These years,the rapid development of stem cell technology has provided new ideas for the source of functional liver cells.On the one hand,stem cells have unlimited selfproliferation capacity,which can meet the needs of the number of cells;on the other hand,stem cells have the potential to differentiate into hepatocytes and bile duct cells,further meeting the needs of cell types.Scientists have explored and optimized the scheme of human embryonic stem cells(ESCs)to induce differentiation into hepatocity-like cells.Coating Matrigel,laminin,fibronectin and other extracellular matrix components can provide an appropriate microenvironment for stem cell differentiation,leading to the improved efficiency of induced differentiation as well as more complete functions of obtaind hepatocyte-like cells.Despite of this,hepatocyte-like cells can still only have part of the liver function and merely reach to the level of fetal liver.If they are to be used in clinical transplantation,they need to be further mature.Extracellular matrix(ECM)is the most important extracellular microenvironment.These macromolecular secreted by resident cells have different spatial distribution characteristics and dynamic changes,which constitute a microenvironment suitable for different tissues and cell types.ECM is a microenvironment with physical,chemical and biological properties.In addition to providing mechanical support and connection for cells,it can also interact with receptors on the cell surface and transmit signals to cells to regulate cell growth,development,maturity,and metabolism,affecting the fate of cells.However,ECM is a complex molecular network system,rather than one or several independent soluble components.Its function depends on the joint effect of multiple molecular networks,which is difficult to replicate through artificial synthesis.The decellularized scaffold obtained after decellularization of natural biological tissue is rich in active molecules secreted by a large number of native primary cells,and its abundance is comparable to that of native tissue.It is the most reliable source of tissue-specific ECM.Based on the above,this research aims to obtain a high-quality rat liver decellularization scaffold(LBS)using the "four-step perfusion method",and the LBS is pulverized and used in the process of hepatic induced differentiation of human embryonic stem cells to promote the maturation of hepatocyte-like cells and improve their function.This research is divided into the following three aspects:1.Preparation and quality evaluation of rat liver acellular scaffoldLBS is prepared through the "four-step perfusion method",that is,in-situ perfusion and decellularization of the rat liver using decellularization solution,concentrated sodium chloride solution,nuclease solution,and saline.The obtained rat LBS has a complete capsule structure with clear veins.Tissue staining,quantification of DNA,collagen and Glycosaminoglycans(Gags)confirm histologically and molecularly that the "four-step perfusion method" can make high-quality LBS,in which the cellular components are thoroughly removed,matrix components such as collagen,Gags and Fibronectin are retained in large quantities.2.hepatic induction of embryonic stem cells based on LBS powderAfter freeze-pulverizing the high-quality LBS obtained by the "four-step perfusion method",it is added as the coating component to the hepatic induction process of human ESCs.Using commercial Matrigel as a control,hepatocyte differentiation is performed.Immunofluorescence staining and real-time fluorescence quantitative PCR are used to detect the expression of lineage markers at various stages of hepatic differentiation.Hepatic cell maturation markers proved that LBS can accelerate the process of ESCs hepatic differentiation,and the phenotype of hepatocyte-like cells is closer to mature hepatocytes.3.Evaluation of the maturity and function of hepatocyte-like cellsRNA of obtained hepatocyte-like cells is extracted to perform real-time fluorescence quantitative PCR to detect the expression of function-related genes,including urea synthesis,albumin synthesis,drug metabolism enzymes,transport proteins,nuclear receptor.Synthesis capacity of urea and albumin and metabolic enzyme activity are detected at the molecular level.The results show that,under the influence of LBS,the aforementioned functions are increased in varying degrees.So far,we have successfully used rat LBS to promote the differentiation of human ESCs into hepatocyte-like cells.We also test the related functions of hepatocyte-like cells.The experimental results,including real-time fluorescent quantitative PCR,immunofluorescence staining and cytochrome enzyme activity detection prove that LBS can promote ESCs induced differentiation into hepatocyte-like cells at a faster speed and a larger proportion.The hepatocyte-like cells have the phenotype closer to mature hepatocytes,whose functions of synthesis and metabolism are also promoted.