Waddington Landscape For Induction Of Hepatocyte-like Cell Formation In Vitr

The liver is one of the most important organs in the human body,responsible for various physiological functions such as metabolism,detoxification,synthesis,and storage.Hepatocyte is the main parenchymal cell of the liver and the primary executor of liver function.Liver diseases seriously affect the quality of life of patients,it is not curable due to the shortage of donor livers.Understanding the regulatory mechanism underlying hepatocyte fate determination will facilitate the differentiation and expansion of hepatocytes in vitro for the treatment of liver diseases.According to Waddington’s epigenetic landscape hypothesis,cells differentiate along a low-energy path from pluripotent cells with high differentiation potential to terminally differentiated cells with low differentiation potential during the developmental process.The entropy of the top landscape represents the differentiation potential of cells,while the regulatory network of the bottom changes the path of the top layer,determines the cell state,and controls the cell fate decision.The methods for generating hepatocytes in vitro include induced differentiation of pluripotent stem cells and transdifferentiation of other cell types.In this study,we profiled a Waddington’s landscape for in vitro induction of hepatocytes by analyzing the entropy changes and transcriptional regulatory networks of the two methods,namely the stepwise induction of embryonic stem cells into hepatocyte-like cells and the transdifferentiation of intrahepatic cholangiocyte organoid(ICO)into hepatocyte-like cells.In the differentiation system from embryonic stem cells to hepatocyte-like cells,key cell types in the system were identified by performing single-cell transcriptome and chromatin accessibility in parallel and analysis on cells at each stage,including embryonic stem cells,definitive endoderm cells,hepatic endoderm cells and hepatocyte-like cells.Entropy analysis showed that the in vitro differentiation of ESC to hepatocyte was consistent with Waddington’s landscape model.We constructed global transcriptional regulatory networks and cell-type-specific transcriptional regulatory networks for the differentiation process,scored the importance of transcription factors according to network topological properties,and illustrate the Waddington’s landscape of this process.The structure and properties of the cell-type-specific regulatory network change continuously during induced differentiation.Through the analysis of the bottom regulatory network,we found that changes in key transcription factors and their target gene functions are similar to the development of hepatocytes in vivo,suggesting that we can use this system to model some aspects of liver development.We also studied the ICO transdifferentiation system by performing transcriptome sequencing on each group of cells in the system.We performed entropy analysis on the three groups in the transdifferentiation system,constructed the transcriptional regulatory network of each group,ranked the importance of transcription factors according to the activity of regulons,and delineated the Waddington’s landscape of the transdifferentiation process.We further compared the transcriptional regulatory networks of the hepatocyte-like cells obtained from two induction differentiation systems and identified common transcription factors and regulatory relationships.This study depicted Waddington’s landscapes of in vitro-induced hepatocyte-like cells,and interpreted the top cell states and bottom transcriptional regulatory networks,providing a foundation for exploring the fate determination process of hepatocyte development.