| Introduction The liver,the largest digestive organ in the body,is composed mainly of hepatocytes and biliary epithelial cells（BECs）,which are essential for liver metabolism,detoxification,and bile modification.Under normal conditions,the liver maintains homeostasis through slow hepatocyte proliferation.However,in cases of acute and chronic liver injury,new hepatocytes and cholangiocytes to aid in liver repair.Unfortunately,some patients with abnormal liver states may experience a sharp decline in their liver regeneration capacity,leading to liver fibrosis and end-stage liver disease（ESLD）,a severe complication of various chronic liver diseases,and its morbidity and mortality rates have been increasing over the past few decades.Understanding the mechanisms underlying liver regeneration and dysfunction is crucial for developing effective treatments for liver diseases.Cell therapy has emerged as a promising approach for end-stage liver disease and is a key component of regenerative medicine.Hepatocytes are the primary parenchymal cells that carry out crucial biological functions in the liver.However,their transplantation has not yet proven to be fully effective in treating liver failure.The discovery of ideal seed cells for clinical transplantation therapy,which are abundant in number and capable of in vivo engraftment while maintaining their function,requires a suitable in vivo model.Our research group has previously shown that primary hepatocyte transplantation effectively treats liver failure in a mouse model with a fumarylacetoacetate hydrolase（Fah）deficiency.This demonstrates the potential of hepatocyte transplantation as a promising therapy for liver diseases.After stopping the administration of NTBC,the host mice experienced rapid liver damage and necrosis.However,injection of exogenous Fah-positive hepatocytes（donor cells）through the spleen resulted in the rapid replacement of host Fah-negative cells by the transplanted cells within 2-3 months,enabling the host mice to metabolize tyrosine and avoid hepatocyte necrosis and liver failure.Moreover,during the xenotransplantation process,the transplanted human hepatocytes have been found to exhibit up to 90%efficacy in engraftment.Therefore,the Fah-deficient mouse model holds great potential for exploring liver regeneration following cell transplantation.Recent advances in single-cell sequencing and functional profiling technologies have enabled the identification of distinct hepatocyte subpopulations with unique gene expression profiles and functional properties.For example,zonation gene expression patterns have been observed in hepatocytes located in different regions of the liver lobule,reflecting their specialized metabolic functions.Further understanding of hepatocyte heterogeneity and its underlying mechanisms is critical for developing effective therapies for liver diseases.Targeting specific hepatocyte subpopulations may enable more precise treatments for liver diseases,while elucidating the molecular mechanisms that regulate hepatocyte diversity and function may reveal novel therapeutic targets.Therefore,the heterogeneity of hepatocytes is a key aspect of liver physiology and pathology.Ongoing research in this area is expected to provide new insights into the regulation of hepatocyte diversity and function,which may lead to the development of more effective therapies for liver diseases.In this study,we utilized single-cell sequencing technology and cell tracking techniques to investigate the changes in the state of transplanted hepatocytes,the characteristics of cell subpopulations,and the underlying mechanisms of liver regeneration following cell transplantation.Our results demonstrate that transplanted cells undergo dynamic changes in their state,and that these changes are closely associated with their functional properties and ability to regenerate the liver.Chapter 1 Single-cell omics reveals dedifferentiation changes of hepatocytes of early post-transplantation stageObjective:Exploring the changes of exogenous hepatocytes colonized in the liver of host Fah-/-mice.Methods:1.We generated Rosa26-LSL-td Tomato transgenic mice on a C57BL/6 background and simultaneously constructed adeno-associated virus 8（AAV8-TBG-Cre）for in vivo transfection via tail vein injection,enabling mature hepatocytes to label red fluorescence.2.We generated Fah gene-deficient mice on a C57BL/6 background and maintained their liver tyrosine metabolism using NTBC-containing drinking water.3.Recipient mice were divided into two groups-control（Sham）and transplantation-and their serum indicators of liver function were assessed at 3,6,9,and 12 weeks post-transplantation.We used RT-PCR and immunofluorescence staining to detect the expression of td Tomato,AFP,H19,Tnfrs12a,Id3,Ki67,MCM2,PCNA,and Ccnd1 in transplanted cells.4.Using flow cytometry sorting of single-cell suspension,we isolated exogenous transplanted cells and their progenies（marked with red fluorescence）in the transplanted mouse liver,which were used to prepare samples for single-cell sequencing.Results:1.Following stringent quality control and standardization,we captured and analyzed28,342 high-quality cells using single-cell sequencing,which were subsequently classified into 13 distinct cell clusters.Differential expression analysis of these clusters revealed two distinct populations（designated as group1 and group2）,with group1being predominantly absent in primary hepatocyte samples but markedly enriched in samples obtained at 3 weeks after transplantation,followed by a gradual decline in the6-week and 12-week samples.These findings were validated by RT-PCR and immunofluorescence staining of liver tissue for Ki67,PCNA,MCM2,and Ccnd1.2.By comparing the expression of liver stem cell markers in the two populations,it was found that the group1 population up-regulated the levels of AFP and H19,and also highly expressed liver regeneration-related gene Tnfrs12a and hepatoblast marker Id3,AFP,H19,Tnfrs12a,Id3 in liver tissue were verified by RT-PCR and immunofluorescence staining.Further comparing the cell entropy value（Cyto TRACE value）,it was found that the group1 population obtained a higher score,indicating that this group of cells may have higher developmental potential.By comparison with classical tertiary biliary reprogramming,transplanted cells highly expressed hepatocyte lineage-related genes and lowly expressed biliary lineage-related genes,and low-level Spp1 and SOX9 expressions were only detected in 3-week-old transplant samples.3.There was no significant difference in the results of serological liver function indexes between the empty vector group and the virus transfection group at different time points;H&E staining showed that the liver lobule structure was clear and closely arranged;The red fluorescence signal intensity of the cells had statistically significant difference（P<0.05）;fluorescent co-staining showed that td Tomato-positive hepatocytes co-expressed with mature hepatocyte markers FAH and HNF4α,but not co-expressed with mature biliary epithelial cells CK19.4.After stopping the drinking water containing NTBC,the mice in the sham group quickly developed arched backs and erect hairs in about 2-3 weeks,and in about 5weeks,90%of the mice in the sham group died,and serological tests showed that transplantation The time change curve of the body weight of the mice showed that the mice in the transplantation group dropped to the lowest point at about 3 weeks,and then gradually recovered to the level before transplantation.Immunohistochemical staining showed that the positive proportion of liver FAH and td Tomato immunohistochemical staining in the transplanted group gradually increasedConclusion:1.Adeno-associated virus can efficiently and safely label mouse mature hepatocytes.Through Flow Cytometry（FCM）,mature hepatocytes can be transplanted and Fah gene-deficient mice can be rescued.2.In the early post-transplantation period,the transplanted cells dedifferentiated,accompanied by the up-regulation of the proliferative ability of the hepatocytes.3.The transplanted hepatocytes maintain the stability of their own lineage during the dedifferentiation process,initiate liver dedifferentiation and promote liver regeneration.Chapter 2 Zonation properties and regularity of transplanted hepatocytes during liver regenerationObjective:To investigate changes in liver zonation characteristics during liver regeneration.Methods:Using a Fah gene deficient mouse model,we conducted single-cell sequencing analysis before and after transplantation at three different time points（3,6,and 12 weeks）to evaluate transplanted cell subpopulations.We performed polychromatic fluorescence staining with partitioning characteristic genes to define liver partitions and observe changes in partition proportions.Results:1.The algorithm developed based on Halpern can analyze the relationship between Cyp2f2,Cyp2e1,Glul,Ass1 and the following cluster analysis shows the correct mutually exclusive distribution.2.Separate cluster analysis of 3 samples at different time points after transplantation showed that mouse hepatocytes at 6 weeks in the middle stage of liver regeneration and 12 weeks in the end stage of liver regeneration were similar to those before transplantation,with complete liver partition and clear partition hierarchy.However,in the early 3 weeks of liver regeneration,a group of cells with ambiguous staging features appeared,expressing both PV and CV features,and upregulating Hamp2,Igfbp2 expressions.From the perspective of spatial distribution of partition coordinates,the partition characteristics gradually deviated from the PV area when the samples from 3-week to 6-week,and recovered to the state before transplantation at the12th week.3.By multicolor fluorescence staining of the zoning characteristic genes,we could observe that the zoning characteristics of the wild-type mice liver were complete.The liver cells near the CV region showed GS+,the liver cells near the PV region showed E-CAD+,and the transition region between the two showed GS-E-CAD-.At 1 week and3 weeks after drug withdrawal,E-CAD+hepatocytes of Fah-/-mice gradually increased and transitioned to the middle region,while liver cells of GS+remained relatively stable.In the transplantation model,labeled td Tomato+mature hepatocytes were evenly distributed in different regions before transplantation.After transplantation,liver cell partitioning characteristics were weakened,and td Tomato+hepatocytes gradually transitioned from PV region to intermediate region,and finally filled CV region and resumed liver partitioning characteristics.Conclusion:1.In the process of liver regeneration,transplanted cells start from the PV region,gradually transition along the middle lobular region to the CV region and eventually fill the whole liver.2.The initial transplanted cells had significant and regular partitioning characteristics,and the partitioning was weakened early after transplantation.At the same time,some hepatocytes without partitioning bias were dedifferentiated and expressed AFP,and finally the partitioning characteristics of liver were restored.3.The AFP+hepatocytes with regional distribution bias are mainly expressed in the PV region,and the driving force of proliferation caused by this is the factor that causes the transplantation cells to selectively proliferate from the PV region and gradually transition to CV.Chapter 3 Elucidating the cellular and molecular mechanisms of liver regeneration following transplanted cell therapyObjective:To explore the mechanism of liver regeneration by transplanted cells and the regulation mechanism of liver cell plasticityMethods:1.Nonnegative matrix factorization（NMF）was used to identify expression programs in cells,and pathway enrichment of different transcriptional programs was performed.2.The intersection of genes identified by NMF transcription program P1 and AFP+cell transcriptome was selected.group1 and group2 were scored using the gene sets after the intersection.3.ROS scores were performed for different samples by GSVA,and key transcription factors were analyzed jointly by ATAC-seq（Chromatin accessibility data）.4.Identify transcription factors（TFS）of cells by using SCENIC and calculate their activities,and construct potential gene regulatory network（GRN）of transplanted cells;5.By comparing the single cell sequencing transcriptome of liver regeneration after transplantation in this study with the single cell transcriptome data of liver resection model,APAP injury model and hepatocellular derived organoids in the open database,the expression patterns and functions of different transcription programs were observed.Results:1.By identifying the expression programs of all samples through NMF,two stable transcriptional programs P1 and P2 can be obtained,which correspond to AFP+dedifferentiated cells and mature liver cells respectively.2.The ROS expression levels of the transplanted cells showed an increasing trend from before transplantation to 3 weeks,and a decreasing trend from 3 weeks to 6 weeks and 12 weeks,and then recovered to the pre-transplantation level.P53 target genes Trp53/p53,Cdkn1a/p16,and Cyp2a4/5 were significantly up-regulated in 3-week samples.According to transcriptional level analysis,the key transcription factor of intracellular antioxidant response was Nfe2l2/Nrf2 and its downstream driving antioxidant genes（Hmox-1/HO-1,GST,GCS,Gclc,Nqo1,including Cyp2a4/5）up-regulated.Lipid accumulation in cells increased significantly 3 weeks after transplantation.The expression of fatty acid synthesis gene（Fasn,Acly,Elovl5）,fatty acid transport gene（Cd36,Fabp4）and fatty acid oxidation gene（Hadh）was up-regulated,and remained stable with the de novo synthesis of fatty acids in AFP+cells.3.Genes positively associated with AFP in P1 program were obtained,and a gene set reflecting the dedifferentiation state was obtained by intersection with genes specifically up-regulated in 3-week samples.SLICE algorithm was used to further prove the accuracy of the gene set defining the dedifferentiation state.4.The samples were scored 3 weeks after transplantation through the gene set of dedifferentiation state.Five new subgroups C1-C5 were obtained by single clustering analysis of AFP+cell populations.5.C3 population activates the transcription factor Nfe2l2/Nrf2.It is observed that the downstream target genes are regulated by proliferator-activated receptors（PPAR）,including Cyp2a4/5.AFP;In addition,C3 also activated Tbx3,Gata6,Foxa1 dry related transcription factors,TGF-βsignaling pathway is also enriched in C3,thus causing the epithelial-mesenchymal transition（EMT）to become the key to early hepatocyte dedifferentiation after transplantation.Conclusion:1.hepatocytes were dedifferentiated and expressed AFP in the early stage after transplantation.Meanwhile,the cell population was enriched in P53,cell cycle and iron death signaling pathways.2.Lipid accumulation and ROS levels of hepatocytes increased significantly in the early stage after transplantation,and the up-regulated P53 pathway was involved in protecting liver function and inhibiting iron death;Nrf2 transcription factor upstream and Cyp2a4/5 downstream are up-regulated to combat oxidative stress. |
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