Lineage Tracing And Single Cell RNA Sequencing Reveals The Change And Mechanism Of Transplanted Hepatocytes During In Vivo Proliferation

Chapter 1 IntroductionOver the past few decades,chronic liver disease has risen inexorably as one of the major global health and economic burdens.Currently,about 844 million people worldwide suffer from liver disease and nearly 2 million died every year,which accounting for 4% of all deaths worldwide.End-stage liver disease is the end-stage of various chronic liver diseases that develops after inflammatory changes in the liver lead to fibrosis and destruction of liver structure and function.Liver transplantation is considered as the gold standard for the treatment of end-stage liver disease and is the only treatment with proven long-term clinical benefits.However,the number of donor livers is insufficient to satisfy the growing clinical demands,and approximately 10%of patients still die every year while waiting for liver transplantation.Hepatocyte transplantation is considered to be an effective alternative to liver transplantation due to its strong replication potential.Especially in the context of metabolic diseases,once implanted in the recipient’s liver,transplanted cells can provide missing or impaired liver function.However,liver cells have obvious heterogeneity,and can be divided into three regions according to their metabolic function: periportal region,pericentral vein region and lobular middle region.In addition to different metabolic functions,hepatocytes in different regions have different hepatogenic abilities in liver homeostasis or in different models of liver injuries.Axin2+ and Lgr5+ hepatocytes around the central venous area,Mfsd2a+Hnf4α+Hnf1β+CK19-and Hnf4α+AFP+Ep CAM+ hepatocytes around the portal vein,and TERTHigh hepatocytes that scattered distribute in the hepatic lobule.All of them are believed to be responsible for liver regeneration under homeostasis and/or certain liver injury conditions.At present,there is a great controversy about the origin of hepatocytes in the process of liver regeneration.However,the prevailing view is that all of the hepatocytes that location in the hepatic lobule own the ability to regenerate.However,as an alternative or transitional treatment for liver transplantation,the origin of new hepatocyte and the cellular mechanism remain unclear during the repopulation of the donor hepatocytes after them transplanted into the diseased host liver.In order to accurately investigate the fate of donor hepatocytes and their progenitors,we plan to analysis the proliferation process of hepatocytes by combined lineage tracing with single-cell RNA sequencing.Chapter 2 Application of Cre-loxp system in hepatocyte tracingObjective: Hepatocytes were fluorescently labeled by viral infection and tested for its safety,efficacy and specificity.Methods: Hepatocytes were labeled by AAV-TBG-CRE virus vector injected into the tail vein of Roas26-LSL-td Tomato mice.Serological liver function indexes(ALB,ALT,AST and TBIL)were detected on the third day after injection.On the 7th day,the liver tissues were collected for H&E staining to observe the liver tissue structure.The validity of fluorescent labeling was verified by cell immunofluorescence,flow cytometry,frozen section and immunohistochemistry.Fluorescence expression in different tissues and organs was detected by small animal IVIS system and immunohistochemistry.Results: We successfully completed fluorescent labeling of hepatocytes by viral infection.There was no significant difference in serological liver function indexes between virus vector group and PBS control group(P > 0.05).H&E staining showed that the liver tissues of the two groups were clear without structure disorder.Macroscopic view,the liver of mice with viral vectors infection was brick red,while the liver of the PBS control group showed light yellow.Nearly 74% of td Tomato+hepatocytes were obtained from the viral vector infection group by flow sorting,while none td Tomato+ hepatocytes were collected from the control group.Cell immunofluorescence confirmed that spontaneous td Tomato fluorescence overlapped with immunostaining fluorescence.Frozen sections and immunohistochemical staining of liver tissue showed that almost all cells showed td Tomato positive.The results of IVIS and immunohistochemical staining showed that the td Tomato expression was only detected in the liver of the mice which infected with the virus.Conclusion: Fluorescence labeling of Rosa26-LSL-td Tomato mouse hepatocyte by AAV8-TBG-Cre virus vector is safe,efficient and specific.Chapter 3 Hepatocyte transplantation rescues the liver function of liver failure model miceObjective: The liver failure mouse model was constructed and the therapeutic ability of hepatocyte transplantation was verified.Methods: The liver failure model was established by Fah gene defect(Fah-/-)mice,and the liver function indexes of the model mice were detected by serology.H&E staining,immunohistochemical staining and serological examination were used to evaluate the efficacy of liver failure after hepatocyte transplantation at different repopulation time points.In addition,the survival status of mice was analyzed by weight curve and survival curve.Results: By withdraw the NTBC from Fah-/-mice drinking water,it was found that the levels of ALB in liver serum of mice were decreased,while ALT,AST and TBIL were increased.H&E staining of liver tissue showed that the original tissue structure of liver was destroyed,vacuolar cells and dead cells appeared after NTBC withdraw.Immunohistochemical staining of FAH protein in recipient liver samples at3,6,9 and 12 weeks after transplantation showed that Fah positive cells were successfully implanted into the host liver,and the proportion of FAH positive area increased with the increase of transplantation time.Serological examination showed that the serum ALB level gradually increased,while ALT,AST and TBIL levels gradually decreased with the increase of repopulated time,indicating the improvement of liver function.Body weight data showed that the body weight of mice decreased gradually in the first 3 to 4 weeks after hepatocyte transplantation,and gradually increased and returned to the initial level after 4 weeks.Survival curve data indicated that the survival rate of mice in hepatocyte transplantation groups was significantly higher than that in control groups(P < 0.05).Conclusion: Hepatocyte transplantation can effectively repopulate the liver of Fah-/-mice,thereby improving liver function and maintaining long-term survival of them.Chapter 4 Single cell RNA sequencing analysis of continuous in vivo repopulation of hepatocytesObjective: Single cell RNA sequencing(scRNA-seqing)was performed to analyze the hepatocytes heterogeneity and the changes of transcription profile after continuous in vivo cell proliferation.Methods: scRNA-seqing samples were prepared by collagenase infusion and flow sorting according to the experimental design process.Cell isolation,cell lysis and m RNA capture were performed by using the Singleron Matrix? single cell processing system;Single cell transcriptome amplification and sequencing library preparation by using EXSCOPE? Single cell RNA Library Kit;Inspection of c DNA library and sequencing library by capillary electrophoresis;Using Cele Scope single-cell software,the sequencing datas were transformed into cell expression matrix;Seurat R software package for preprocessing and normalization;PCA dimension reduction analysis,UMAP clustering analysis,differential expression analysis and pseudotime analysis were performed on the sample data of different transplant rounds and repopulation time points.Results: The cell activity was above 80% and the single-cell rate was above90%.The results of capillary electrophoresis showed that the quality of c DNA library and sequencing library met the standard of second-generation sequencing.After data screening,a total of 5.6×104 cells were obtained,And the average reads and UMI was4.8×104 and 1.1×104,respectively.The average number of genes detected in each sample was 2.1×104 and 1500 genes detected in per cell.The samples had an average saturation of 56%.PCA dimensionality reduction analysis and UMAP clustering analysis showed that the first round of hepatocyte transplantation samples could be divided into 11,13 and 11 cell clusters,respectively,and the second round of hepatocyte transplantation samples were clustered into 15,12 and 12 cell clusters,respectively.Cell subpopulations expressing specific genes were found in different transplanted cycles and repopulation time points.Such as,cells expressing macrophage-related Saa family genes,cells expressing Ube2 c and Stmn1 genes which related to cell cycle and mitosis,and cells expressing Tmsb4 x genes which related to cell proliferation and differentiation,and cells expressing hepatic stem gene AFP,and cells expressing cytochrome P450 gene,etc.Differential gene expression heat maps also confirmed the differential expression of each cell subpopulation on the transcription spectrum.Monocle2 conducted a pseudotime analysis of hepatocyte clusters,and the results showed that the samples at different repopulation time points showed two distinct evolutionary branches,which proved that there were developmental differences among each cell subpopulation perhaps.Conclusion: Hepatocytes exist obvious heterogeneity,which may change undergoing serial repopulation.Chapter 5 Role of AFP positive hepatocytes during continuous in vivo repopulationObjective: scRNA-seqing data were used to analyze the continuous in vivo repopulation process of the transplanted hepatocytes.Methods: scRNA-seqing sample data were integrated for analysis.Firstly,Beer algorithm was used to remove the batch effect of the samples,and then UMAP algorithm was used to reduce the dimension of the comprehensive data set.Hepatocyte indexes of all cell samples were compared by GSVA enrichment analysis.UMAP has exhibited the clusters and the zonation of the hepatocytes.SLICE algorithm calculates the entropy of single cell.The co-expression patterns were analyzed by GSVA enrichment analysis and non-negative matrix decomposition algorithm.RNA velocity analysis predicts cell state and differentiation tendency.Double immunofluorescence staining was used to detect AFP positive cells.Scenic algorithm is used to analyze the transcriptional regulatory network of a particular cell subpopulation.Results: The proportion of td Tomato+ cells in all samples ranged from 72.68%to 99.7%,and the proportion of td Tomato+ cells in primary hepatocytes was the highest,and the lowest in early repopulation time point.GSVA analysis showed that the hepatocyte index was lower in the third week time point than that at other time points.UMAP clustering visualization results showed that 40546 td Tomato+hepatocytes from different repopulation time points could be clustered into 16 cluster subgroups.Cluster 6,Cluster 8,Cluster 11 and Cluster 12 were the main subpopulations of hepatocytes at the third week time point.Cell cycle scores showed that most cells in cluster 6 were inside the cell cycle,and the cycling proportion of the cells in other clusters were basically similar.The graph of proliferative gene expression shows that the expression in cluster 6 is the highest.Cluster analysis showed that hepatocytes had obvious zonation specificity,but the Zonation characteristics became blurred at the most active stage of proliferation.The SLICE algorithm calculated the sc Entropy value and found that the sc Entropy of the sample after third week repopulation was the highest,which was consistent with its expression of hepatic stem cell markers.Both GSVA enrichment analysis and NMF algorithm clustered the 16 clusters into 5 programs,and the cells expressing program were mainly from the early recolonization samples.UMAP clustering showed that AFP+ hepatocyte population was highly coincident with progrom3 expression.RNA velocity analysis suggested that AFP+ hepatocyte population played a pivotal role in cell proliferation.Double immunofluorescence staining confirmed the presence of AFP+ cells in the repopulation colony.Analysis of transcription factor regulatory network showed that E2f4,Ybx1,Atf3,Pole4 and Bcl3 were involved in transcription regulation of AFP+ hepatocytes.Among them,transcription factor E2f4 may play the most regulatory role.Conclusion: AFP+ hepatocyte population is produced during continuous in vivo repopulation,and AFP+ hepatocyte perhaps plays a key role in the repopulation after hepatocytes transplantation.