Growth pattern and phenotype identification of hepatic stem cells from mice

Bone Marrow–Derived Hepatic Oval Cells Differentiate Into Hepatocytes in 2-Acetylaminofluorene/Partial Hepatectomy–Induced Liver Regeneration

Isolation and Expansion of Hepatic Stem-like Cells from a Healthy Rat Liver and their Efficient Hepatic Differentiation of under Well-defined Vivo Hepatic like Microenvironment in a Multiwell Bioreactor

Objective Epithelial-mesenchymal transition （EMT） has a role in the proliferation and metastasis of various types of cells. This study investigates the hepatic oval cellts mechanism of EMT induced by histone deacetyiase inhibition and the resulting cell motility increase from EMT. Methods Hepatic oval cell stem cell markers and marker changes were detected by flow cytometry, and after histone deacetylase inhibition induced EMT, the morphological changes were recorded. Western blot and quantitative real-time polymerase chain reaction detected the expression of E-cadher- in, vimentin and Snail. Furthermore, confocal microscopy analysis recognized the nuclear translocation of Snail. Results Flow cytometry revealed no changes in the stem cell properties of hepatic oval cells in the cell culture process. Oval cell EMT, induced by HDACi, was observed through morphological changes, a reduction of the epithelial cell marker E-cadherin, and an increase of the mesenchymal cell marker Vimentin. HDACi can promote the expression and nuclear translocation of Snail, which is the key hepatic oval cell transcription factor for both EMT and enhanced motility. Therefore, Snail RNA interference can suppress HDACi induced EMT in hepatic oval cells. Conclusions In conclusion, histone deacetylase inhibition induces hepatic oval cell epithelial-mesenchymal transition by Snail. Key words: Hepatic oval cell; HDACi; Epithelial-mesenchymal transition; EMT

The liver is an organ with tremendous regenerative capacity. Hepatic stem cells, identified by flow cytometric sorting and single cell-based assays as c-Met (+)CD49f (+/low)c-Kit (-)CD45 (-)TER119 (-) cells in murine fetal livers, possess multilineage differentiation potential and self-renewing capability. These cells can differentiate in vitro as well as in vivo into some epithelia capable of reconstituting tissues within the liver, pancreas, and intestine following appropriate transplantation. The isolated fetal hepatic stem cells are also characterized by the ability to maintain their self-renewal capability in the developing liver. Thus, these cells may be ideal vehicles for delivering therapeutic genes to the liver. We also examined the relationship between fetal hepatic stem cells and hepatic oval cells in the adult liver. These two subtypes shared both multipotential capabilities of differentiation and surface marker expression profiles, suggesting that hepatic oval cells are probably the progeny of fetal hepatic stem cells. We also address the related topics of hepatocyte repopulation, exogenous hepatic stem cells, and transdifferentiation. Topics ranging from the fundamental properties and comprehensive descriptions of hepatic stem cells to current progress in the field are discussed in this brief article and our recent studies are addressed.

Mesenchymal−epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal−epithelial transition associated with stem cells is unclear. To determine whether mesenchymal−epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein.ConclusionHepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal−epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.

IntroductionChimeric mice with humanized livers were recently established by transplanting human hepatocytes. This mouse model that is repopulated with functional human hepatocytes could be a useful tool for investigating human hepatic cell biology and drug metabolism and for other preclinical applications. Successfully transplanting human hepatocytes into mice requires that recipient mice with liver failure do not reject these human cells and provide a suitable microenvironment (supportive niche) to promote human donor cell expansion and differentiation. To overcome the limitations of current mouse models, we used Alb-TRECK/SCID mice for in vivo human immature hepatocyte differentiation and humanized liver generation.Methods1.5 μg/kg diphtheria toxin was administrated into 8-week-old Alb-TRECK/SCID mice, and the degree of liver damage was assessed by serum aspartate aminotransferase activity levels. Forty-eight hours later, mice livers were sampled for histological analyses, and the human donor cells were then transplanted into mice livers on the same day. Chimeric rate and survival rate after cell transplantation was evaluated. Expressions of human hepatic-related genes were detected. A human albumin enzyme-linked immunosorbent assay was performed after 50 days of transplantation. On day 60 after transplantation, drug metabolism was examined in mice.ResultsBoth human primary fetal liver cells and hepatic stem cells were successfully repopulated in the livers of Alb-TRECK/SCID mice that developed lethal fulminant hepatic failure after administering diphtheria toxin; the repopulation rate in some mice was nearly 100%. Compared with human primary fetal liver cells, human hepatic stem cell transplantation rescued Alb-TRECK/SCID mice with lethal fulminant hepatic failure, and human hepatic stem cell-derived humanized livers secreted more human albumin into mouse sera and also functioned as a “human liver” that could metabolize the drugs ketoprofen and debrisoquine.ConclusionOur model of a humanized liver in Alb-TRECK/SCID mice may provide for functional applications such as drug metabolism, drug to drug interactions, and promote other in vivo and in vitro studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0038-9) contains supplementary material, which is available to authorized users.

Although it was proposed almost 60 years ago that the adult mammalian liver contains hepatic stem cells, this issue remains controversial. Part of the problem is that no specific marker gene unique to the adult hepatic stem cell has yet been identified, and regeneration of the liver after acute injury is achieved through proliferation of adult hepatocytes and does not require activation or proliferation of stem cells. Also, there are differences in the expected properties of stem versus progenitor cells, and we attempt to use specific criteria to distinguish between these cell types. We review the evidence for each of these cell types in the adult versus embryonic/fetal liver, where tissue-specific stem cells are known to exist and to be involved in organ development. This review is limited to studies directed toward identification of hepatic epithelial stem cells and does not address the controversial issue of whether stem cells derived from the bone marrow have hepatocytic potential, a topic that has been covered extensively in other recent reviews.

Mast cells in the liver.

Integrative Transcriptomic Profiling Reveals Slinically Relevant Stem Cell-Like Subtypes in Early-Stage Resectable Hepatocellular Carcinoma

Objective To investigate the effect of platelet activating factor on the proliferation of cultured hepatic stellate cells in vitro. Methods LX-2 cell line was chosen as the study model of the activated hepatic stellate cells. MTT colo- rimetric assay was used to evaluate the proliferation of HSCs. Cell cycle was evaluated by ways of flow cytometry with PI staining and BrdU incorporation.Results OD_(492) value of PAF groups at concentration from (1 × 10~(-8)-1 × 10~(-3) mol/L), were higher than that of control group (P < 0.05). OD_(492) value of PAF (1×10~(-6)) + PAF-RA × 1 (10~(-7)-1 × 10~(-4)mol/L) groups was less than that of PAF (1 ×10~(-6)mol/L) group (P<0.05). S% and PrI of PAF groups increased significantly. Conclusion PAF can increase the proliferation of hepatic stellate cells in a dose-dependent manner while PAF receptor antagonist reduces the proliferation of hepatic stellate cells induced by PAF. Key words: Platelet activating factor; Hepatic stelate cell; Proliferation

Objective To compare the advantages and disadvantages of the three isolation regents for isolating primary hepatic stellate cells(HSCs) of mice.Methods The mice HSCs was isolated by in situ perfusion of per-perfusion solution,perfusion with collagenase Ⅳ.After digestion,Ficoll-Paque PLUS,perco11 and Nycodenz were use for the gradient centrifugation.To calculate the quantity,purity and the viability of the HSCs.The autofluorescence and Oil O staining were used to identify primary HSCs.Results The quantity of HSCs was (1.0 ± 0.5) × 106,(1.0 ± 0.5) × 105,(0.8 ± 0.5) × 106/ g_liver,respectively.Using Ficoll-Paque PLUS,percoll and Nycodenz,the purity of primary HSCs were 75％-85％,91％-95％,91％-95％,respectively.The viability were all above 90％.Conclusion compared with Ficoll-Paque PLUS and percoll,the Nycodenz is the the best separating medium for isolation and purification of mice primary HSCs. Key words: Liver cirrhosis; Cells, cultured; Cells/isolation and purification; Mice

To determine the treatment effects of transplanted hepatic progenitor cells (WB-F344 cells) combined with heparin on the acute liver injury in SD rats. A total of 2*10(7) hepatic stem cells (WB-F344) infected with GFP lentivirus and 8 μL heparin were transplanted through the spleen in SD rats with acute liver injury, which was induced by an intraperitoneal injection of CCl4. The liver and spleen tissues underwent fluorescence examination 1 day after the transplantation. The liver functions were tested, and the liver tissues were histopathologically examined on the 3rd, 7th, 14th, and 28th day of the cell transplantation. The transfected WB-F344 cells expressed GFP 3 days after the lentivirus infection and were found in the rat liver 1 day after the WB-F344 transplantation. The liver function and histopathological recovery of the liver tissues in the group of WB-F344 transplantation were better than those of the control group (P<0.05). Transplantation of hepatic stem cells combined with heparin can promote the liver recovery in rats with acute liver injury induced by CCl4.

Objective Accumulating reports have suggested that hepatic stellate cells (HSCs) exhibit immunosuppressive ability and may be responsible for the occurrence and development of hepatocellular carcinoma (HCC).The mechanisms through which HSCs affect T-cell-induced adaptive immune responses and the relationship with the regulatory T cells (Treg cells) were studied.Methods We isolated HSCs from wildtype mice to demonstrate the influence of HSCs on T-cell proliferation and explored their effect on Treg cells through mixed leukocyte reactions (MLRs) in vitro.Results We found that activated HSCs could induce T-cell hyporesponsiveness in adaptive immune response by inhibiting the proliferation of T cells andincreasing the quantity of Treg cells.Conclusion Activated HSCs may lead to hypoergia of T cells in adaptive immune reaction and up-regulate the expression of Treg cells,thus facilitating immunotolarance. Key words: Hepatic stellate cell; T cell; Regulatory T cells

Hepatic Stem Cells and Hepatoblasts: Identification, Isolation, and Ex Vivo Maintenance

Human liver development commences during the third to fourth week of gestation. Endodermal cells give rise to hepatic cords representing liver progenitor cells. These progenitor cells are pluripotent because they differentiate along both the hepatocyte lineage (hepatoblasts) and the biliary epithelial cell lineage (ductal plate cells). In addition, ductal plate cells give rise to peri-biliary glands and in some cases to ectopic exocrine pancreatic tissue. Thus, the progenitor cells in the human fetal liver appear to have a broad differentiation potential similar to the putative hepatic stem cells or oval cells in the rat liver, which may develop into hepatocytes, biliary epithelial cells, pancreatic cells, and intestinal cells. Over the last decade, the existence and location of hepatic stem cells has been hotly debated and therefore hepatic development and the pluripotential capacities of liver progenitor cells have generated much interest. This chapter concisely presents the current stage of understanding of human liver development.