Human liver growth and development

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

This Month in Gastroenterology

Inhibition of adult liver progenitor (oval) cell growth and viability by an agonist of the peroxisome proliferator activated receptor (PPAR) family member γ, but not α or δ

Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based invitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations.

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 stem cell nature of normal liver progenitor cells (LPCs) is addressed by studies of normal LPCs in culture. Several questions are addressed such as: What are the patterns of proliferation, lineage commitment, differentiated gene expression, plasticity, and responses to epigenetic and environmental signals? Early studies were interpreted to show that propagable LPCs were derived from dedifferentiated or retrodifferentiated mature liver cells. The recognition that oval cells seen in hepatocarcinogenesis models in rats had characteristics of LPCs suggested that these cells might actually be LPCs or descendants of LPCs. A comparison of more than 30 publications over three decades reporting explants; clonal lines; fresh isolates or strains of cells from noncarcinogen-exposed normal mouse, rat, pig, and human liver; or embryonic tissues indicates that small, immature LPCs, which have the plasticity to mature into ductal cells or hepatocytes, can be obtained from embryonic and fetal tissues, as well as adult liver. A wide variation in the methods of isolation, culture media, feeder layers, growth factors, and substrata used to study putative LPCs in vitro makes comparisons of results from different laboratories difficult. Although the liver is endodermally derived, putative coexpression of primitive hematopoietic and hepatocytic markers is consistent with LPCs in hepatic as well as in blood-forming tissues. In addition to bile duct and hepatocytic differentiation, LPCs have been reported to express markers of pancreatic and endothelial cells in vitro, and to differentiate into bile ducts, hepatocytes, pancreatic islet and acinar epithelial cells, intestinal epithelial cells, and cardiac myocytes after transplantation in vivo. Culture of LPCs on STO embryonic fibroblast feeder layers maintains primitive phenotypes, but requirements of feeder layers appear not to be absolute and are poorly understood. Emerging trends suggest HGF, Flt-3 ligand, SCF, EGF, and DMSO promote hepatocyte differentiation of LPCs, and that transforming growth factorβ, Na+-butyrate, and culture on Matrigel promote biliary differentiation; however, exceptions have been reported. Critical studies on proliferation kinetics have not convincingly shown self-renewal and asymmetric cell division expected of tissue stem cells, but long-term doublings (up to 150 generations) without spontaneous transformation suggest considerable growth potential. The source of LPCs in normal liver remains unknown and controversial. LPCs may be derived from a liver tissue progenitor cell located in the duct or periductal tissue; from retrodifferentiation of more mature hepatocytes; from bone marrow-derived cells, which circulate through the liver; or from bone marrow remnants of intrahepatic embryonic development. Given the lack of well-defined markers for LPCs, incomplete knowledge of their growth characteristics and regulation signals, their apparent heterogeneity, their apparent plasticity, the possibilities of transdifferentiation or retrodifferentiation of other cells to LPCs, or fusion of LPCs with other cells, as well as their potential for tumorigenesis, much research needs to be conducted to understand what LPCs are and how to use them.

Background and AimsmicroRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. miRNAs have been implicated in regulating gene expression in embryonic developmental processes, including proliferation and differentiation. The liver is a multifunctional organ, which undergoes rapid changes during the developmental period and relies on tightly-regulated gene expression. Little is known regarding the complex expression patterns of both mRNAs and miRNAs during the early stages of human liver development, and the role of miRNAs in the regulation of this process has not been studied. The aim of this work was to study the impact of miRNAs on gene expression during early human liver development.MethodsGlobal gene and miRNA expression were profiled in adult and in 9–12w human embryonic livers, using high-density microarrays and quantitative RT-PCR.ResultsEmbryonic liver samples exhibited a gene expression profile that differentiated upon progression in the developmental process, and revealed multiple regulated genes. miRNA expression profiling revealed four major expression patterns that correlated with the known function of regulated miRNAs. Comparison of the expression of the most regulated miRNAs to that of their putative targets using a novel algorithm revealed a significant anti-correlation for several miRNAs, and identified the most active miRNAs in embryonic and in adult liver. Furthermore, our algorithm facilitated the identification of TGFβ-R1 as a novel target gene of let-7.ConclusionsOur results uncover multiple regulated miRNAs and genes throughout human liver development, and our algorithm assists in identification of novel miRNA targets with potential roles in liver development.

Background Understanding the expression of non-coding RNA in the liver during embryonic development provides important insights into liver diseases. Therefore, we investigated circular RNA (circRNA) roles in human liver development, an unexplored research domain. Methods Using high-throughput sequencing and bioinformatics, we analysed foetal liver samples across developmental stages (7–20 weeks post-conception). Differentially expressed (DE) genes were identified and subjected to enrichment analysis using Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG), and Disease Ontology (DO). Modular analysis was performed using the Search Tool for Retrieval of Interacting Genes (STRING), followed by construction of a protein-protein interaction (PPI) network using Cytoscape software. The key genes were screened using Molecular Complex Detection (MCODE). The mRNA levels of hub genes were validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results There were 645 DE circRNAs and 5,145 DE mRNAs between human livers at the three growth stages (HB, EH, and LH). It was found that the activity of circRNAs was boosted remarkably in the hepatoblastic stage. Enrichment analysis found they mainly involved in nervous system regulation of liver function, embryonic organ development and digestive system development. In addition, DE circRNAs were primarily involved in the PI3K-AKT, MAPK and calcium pathways, potentially contributing to adult liver diseases. Notably, only hsa_circ_001471 and novel_circ_017382 were simultaneously identified at all stages and were persistently downregulated. A co-expression regulatory network involving these circRNAs was established. Three hub genes (LGR5, FOXL1 and RSPO3) were identified from the PPI network of 167 genes and may play key roles in human liver development. The RT-qPCR validation results were in agreement with the sequencing data. Conclusions Our findings provide the first insights into the roles and regulatory networks of circRNAs in human liver development, laying the groundwork for further investigations of molecular and signalling networks.

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.

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.

Objective To study the proliferation and differentiation patterns of hepatic stem cells from mice cultured in vitro isolated identify their characteristics.Methods Mice were divided into 5 groups according to the pregnant (embryo days,ED13,ED15 and ED18) or born age (day0 and 3).Hepatic stem cells were isolated and cultured in vitro.The amount of the stem cells as well as their growing situation and differentiation pattern were observed and compared among different groups and markers of stem cells (CD117,CD90.1,CD49f,c-Met),hepatic cells(AFP) were used to identify the cultures.Resulls The cells with best situation of growing as colonies were obtained from ED15 group.Their expression of specific markers suggested that they were hepatic stem cells.The stem cells isolated from ED15 mice in subculturing proliferated in line pattern and differentiated in reverse line pattern.The expression of AFP varied as normal distribution as cell differentiation development.Conclusion Most cells have characteristics of hepatic stem cells isolated from fetal liver and the number of these cells decreases gradually as embryo duration prolongs.The hepatic stem cells proliferate in line pattern and differentiate into hepatic cells after in vitro culture. Key words: Hepatic stem cell; Proliferation; Differentiation; Identification

Hepatic oval cells, progenitor cells in the liver, can differentiate into hepatocytes and bile duct cells both in vitro and in vivo. Although hepatic stellate cells are another important cell component in the liver, less attention has been focused on the relationship between hepatic oval cells and hepatic stellate cells. Hepatic oval cells were isolated from rats fed a choline-deficient diet supplemented with 0.1% ethionine for 6 weeks and characterized by electron microscopy, flow cytometry, reverse transcription polymerase chain reaction, Western blot and bi-direction differentiation. After treatment with transforming growth factor-beta1 (TGF-beta1), changes in cell viability, morphology, extracellular matrix (ECM) expression and immune phenotype were analysed in these cultured and adherent hepatic oval cells. The primary cultured hepatic oval cells were positive for the oval cell-specific markers OV-6, BD-1/BD-2 and M2PK as well as the hepatocyte markers albumin and alpha-foetoprotein. These hepatic oval cells differentiated bipotentially into hepatocytes or bile duct-like cells under appropriate conditions. It is noteworthy that these bipotential hepatic oval cells expressed ECM genes stably, including collagens, matrix metalloproteinases and tissue inhibitor of mellatoproteinase. Furthermore, except for growth inhibition and morphological changes in the hepatic oval cells after exposure to TGF-beta1, there was an increased expression of ECM genes, the onset expression of snail and loss expression of E-cadherin. During this process, TGF-beta1 treatment induced an upregulation of marker genes for hepatic stellate cells in hepatic oval cells, such as desmin and GFAP. Except for the expression of ECM, the cultured hepatic oval cells could induce an increased expression of hepatic stellate cell markers by TGF-beta1 through an epithelial-mesenchymal transition process, which might indicate the contribution of hepatic oval cells to liver fibrosis.

Introduction: There has been a heavy reliance on the use of hepatoma cell
\nlines to study the pathways of hepatocarcinogenesis. Cell lines are transformed, immortalized, and do not mimic the natural pathobiological
\ncharacteristics of hepatocellular carcinoma (HCC).
\nAim: This study aims to develop novel techniques of deriving and culturing
\nprimary dysplastic hepatocytes (DHs) and HCC cells for interventional
\nand functional studies. 
\nMethods: Primary hepatocytes (PHs) are derived from naïve male mice
\nusing the hepatic portal vein perfusion method; primary DHs and HCCs
\nfrom diethylnitrosamine-injected mice are isolated via fluorescence activated cell sorting and mechanical disruption. Cell viability is determined using MTT assay and protein expression by immunoblotting. Cell cycle phase analysis is performed using propidium iodide staining followed by fluorescence activated cell sorting, while p53 and mdm2 chemical inhibition with pifithrin-α and Nutlin-3 are used, respectively.
\nResults: While the viability of PHs is limited to 5 days on collagen-coated plates, primary DHs can be maintained in culture for up to 4 weeks with minimal proliferative activity. In contrast, primary HCC cells undergo active hepatocyte proliferation, evident by several passages and positive proliferating cell nuclear antigen expression. Cell cycle phase analyses confirm that majority of PHs are maintained in cell cycle arrest phase (G0/G1) with only 2 ± 0.02% cells in G2/M phase. However, sorted DHs
\ndisplay an augmented population of G2/M cells (30 ± 0.03%) and such
\npopulations are enhanced in primary HCCs (56 ± 0.1%). Pifithrin-α administration results in decreased p53 mRNA in HCC cells but elicits no
\nchanges in DHs. puma mRNA is decreased in both cell types. Inhibition
\nof p53 exhibits increased mdm2 and downregulation of usp18 mRNA
\nexpression in both cell types. Nutlin-3 treatment suppresses mdm2 mRNA
\nonly in DHs but not HCCs. p53 and usp18 mRNA are downregulated in
\nboth cell types.
\nConclusion: Isolated murine dysplastic hepatocytes exhibit distinct molecular mechanisms/properties compared to HCC cells and may prove to be an appropriate in vitro resource to elucidate the molecular changes early in hepatocarcinogenesis.

Mast cells in the liver.

Lymphotoxin-beta (LTbeta) is a proinflammatory cytokine and a member of the tumor necrosis factor (TNF) superfamily known for its role in mediating lymph node development and homeostasis. Our recent studies suggest a role for LTbeta in mediating the pathogenesis of human chronic liver disease. We hypothesize that LTbeta co-ordinates the wound healing response in liver injury via direct effects on hepatic stellate cells. This study used the choline-deficient, ethionine-supplemented (CDE) dietary model of chronic liver injury, which induces inflammation, liver progenitor cell proliferation, and portal fibrosis, to assess (1) the cellular expression of LTbeta, and (2) the role of LTbeta receptor (LTbetaR) in mediating wound healing, in LTbetaR(-/-) versus wild-type mice. In addition, primary isolates of hepatic stellate cells were treated with LTbetaR-ligands LTbeta and LTbeta-related inducible ligand competing for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT), and mediators of hepatic stellate cell function and fibrogenesis were assessed. LTbeta was localized to progenitor cells immediately adjacent to activated hepatic stellate cells in the periportal region of the liver in wild-type mice fed the CDE diet. LTbetaR(-/-) mice fed the CDE diet showed significantly reduced fibrosis and a dysregulated immune response. LTbetaR was demonstrated on isolated hepatic stellate cells, which when stimulated by LTbeta and LIGHT, activated the nuclear factor kappa B (NF-kappaB) signaling pathway. Neither LTbeta nor LIGHT had any effect on alpha-smooth muscle actin, tissue inhibitor of metalloproteinase 1, transforming growth factor beta, or procollagen alpha(1)(I) expression; however, leukocyte recruitment-associated factors intercellular adhesion molecule 1 and regulated upon activation T cells expressed and secreted (RANTES) were markedly up-regulated. RANTES caused the chemotaxis of a liver progenitor cell line expressing CCR5. This study suggests that LTbetaR on hepatic stellate cells may be involved in paracrine signaling with nearby LTbeta-expressing liver progenitor cells mediating recruitment of progenitor cells, hepatic stellate cells, and leukocytes required for wound healing and regeneration during chronic liver injury.