Identification Of Authentic Liver Stem Cells And Their Differentiation Lineage In Adult Mouse

Tissue stem cells play a critical role in the homeostasis of mammals in vivo. However, liver as one of the most important internal digestive organs, the fundamental problem in the field that how adult liver stem cells participate in turnover and repair of liver cells is still unsolved, even the existence of liver stem cells is controversial. The main problems on research of adult liver stem cells are as follows:first, owning to the strong replication capacity, mature hepatocytes themselves could restore the liver damages via self-renew when liver suffers mechanical injuries or heptectomy; besides, the restoration of hepatocyte is not restricted into a one-time injury, for that it is demonstrated that the rat liver could recover itself to the primitive bulk when received twelve consecutive hepatectomy. Second, livers, similar to muscle tissues and nerve tissues, belong to tissues with a slowly-turnover rate, in that the lifespan of hepatocytes in mice could be.300 days under a physiological conditon. Therefore to clarity the contribution of liver stem cells requires long time span; in addition, mice could have been into aging process as these 300 days passed, so whether liver stem cells would lose the ability of contribution to the liver during the aging remains unknown. Third, liver stem cells and biliary epithelial cells cannot be strictly distinguished between each other based on the markers already known, which increases the difficulty and the risk of misdirection when liver stem cells are traced; it is even recognized that markers of Lgr5 and CD 133 may be lost during stem cells activation, resulting in losing contact with target cells. Forth, distinct laboratories differ in the research models of liver stem cells; for examples, models of acute injuries include 2/3 partial hepatectomy, carbon tetrachloride injection and biliary ligation, while models of chronic injuries include DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet, CDE (choline-deficient diet supplemented with ethionine) consumption, hepatic fibrosis, et al; the discrepancies within different models directly result in differences responding to distinct injuries, which undoubtedly increases the complexity for liver stem cell research. Fifth, "ductular reaction" can be observed among many human liver diseases, that is, many "bile duct-like" cells emerge within liver parenchyma; it remains unknown what these cells are as well as their biological roles. In conclusion, the problems of lacking of specific markers for liver stem cells, discrepancies within different injury models, the damage extent of liver parenchyma, the link between liver stem cells and human liver diseases have constituted the core research in this field. Our study is focusing on the fundamental problem in this field of "what exactly liver stem cells are?", and we are engaged to unmask the mystery of liver stem cells, then to provide the clues and lay the foundation for further clinical application study of liver stem cells.In this study, we try to solve the problem by performing single-cell RNA-squencing on various cell types in the liver. First, we build a Cre/LoxP system for lineage tracing and injury models in mice. Based on evidence that liver stem cells expressing Krtl9, we used Krtl9CreERT/Rosa26R-GFP lineage tracing mouse at 8 weeks-old and injected them with 4mg tamoxifen via intraperitoneal. Then GFP expression was detected after induction for 2 days, followed by liver injury treatment; mice received acute liver injury treatment after tamoxifen induction for 2 days; lul/g body weight for carbon tetrachloride via intraperitoneal injection; chronic liver injury:mice were fed with normal chow diet containing 1% DDC (w/w) for 3 weeks. Secondly, cDNA libraries construction, deep sequencing and transcriptome analysis. After GFP positive cells were sorted from mice liver by FACS, individual cell cDNA libraries was constructed; preliminary identification of cell identity by PCR via markers already known; then took candidate cells for deep sequencing; sequencing data was mapped to mus musculus genome assembly version 9 (mm9) then followed by unsupervised hierarchical clustering analysis, WGCNA and differential expression analysis.3) Clustering the candidate cells, mapping the differentiation lineage of liver stem cells, screening out candidate surface markers and activation related factors for liver stem cells.4) Performing experiments to prove the results.The results from this study are as follows:1) Krt19 lineage cells were acquired from Krtl9CreERT/Rosa26R-GFP tracing mice, which could mark cholangiocytic cells, hepatic cells and potential liver stem cells.2) Selections for 1094 single-cells, constructions for cDNA libraries of each cell, then deep sequencing and transcriptome analysis for 39 candidate cells after PCR identification.3) The acquired cells could be classified into 6 populations according to single-cell transcriptome analysis, which contained 2 types of liver stem cells including quiescent stem cells, activated stem cells, and hepatic progenitor, immature hepatocyte, mature hepatocytes, cholangiocytes.4) Reconstructions of differentiation lineage of liver stem cells according to transcriptome features of distinct cell groups, delineation of molecular signatures for distinct cell groups, screening out specific surface markers of CD63 and CD56 for liver stem cells, and critical pathways activating quiescent liver stem cells, i.e. VEGF and MAPK.5) CD63 was demonstrated as a novel surface marker for authentic liver stem cells via immunostaining in situ, culture in vitro, cell transplantation and lineage tracing in vivo.6) CD56 was regarded to distinguish between activated and quiescent liver stem cells:CD63+CD56+cells were activating liver stem cells, while CD63+CD56-cells were quiescent liver stem cells.7) Growth factors bFGF and VEGF could drive quiescent liver stem cells into activated state.In conclusion, we have described differentiation lineage of liver stem cells via single-cell transcription analysis in mice liver, identified the molecular signature of distinct liver cell groups in mice, demonstrated that CD63 was a specific surface marker for liver stem cells, that CD56 was a surface marker for activated liver stem cells, and that bFGF and VEGF could activate CD63+CD56- quiescent liver stem cells. This study provides a novel thought and method for adult tissue stem cells research, opens up a new research direction for liver stem cells and meanwhile lays a great foundation for human liver stem cells research.