Differential Modulation of miR-122 Transcription by TGFß1/BMP6: Implications for Nonresolving Inflammation and Hepatocarcinogenesis.

Chronic inflammation is widely recognized as a significant factor that promotes and worsens the development of malignancies, including hepatocellular carcinoma. This study aimed to explore the potential role of microRNAs in inflammation-associated nonresolving hepatocarcinogenesis. By conducting a comprehensive analysis of altered microRNAs in animal models with liver cancer of various etiologies, we identified miR-122 as the most significantly downregulated microRNA in the liver of animals with inflammation-associated liver cancer. Although previous research has indicated the importance of miR-122 in maintaining hepatocyte function, its specific role as either the trigger or the consequence of underlying diseases remains unclear. Through extensive analysis of animals and in vitro models, we have successfully demonstrated that miR-122 transcription is differentially regulated by the immunoregulatory cytokines, by the transforming growth factor-beta 1 (TGFß1), and the bone morphogenetic protein-6 (BMP6). Furthermore, we presented convincing evidence directly linking reduced miR-122 transcription to inflammation and in chronic liver diseases. The results of this study strongly suggest that prolonged activation of pro-inflammatory signaling pathways, leading to disruption of cytokine-mediated regulation of miR-122, may significantly contribute to the onset and exacerbation of chronic liver disease.

Extracellular Vesicles as Markers of Liver Function: Optimized Workflow for Biomarker Identification in Liver Disease.

Liver diseases represent a significant global health burden, necessitating the development of reliable biomarkers for early detection, prognosis, and therapeutic monitoring. Extracellular vesicles (EVs) have emerged as promising candidates for liver disease biomarkers due to their unique cargo composition, stability, and accessibility in various biological fluids. In this study, we present an optimized workflow for the identification of EVs-based biomarkers in liver disease, encompassing EVs isolation, characterization, cargo analysis, and biomarker validation. Here we show that the levels of microRNAs miR-10a, miR-21, miR-142-3p, miR-150, and miR-223 were different among EVs isolated from patients with nonalcoholic fatty liver disease and autoimmune hepatitis. In addition, IL2, IL8, and interferon-gamma were found to be increased in EVs isolated from patients with cholangiocarcinoma compared with healthy controls. By implementing this optimized workflow, researchers and clinicians can improve the identification and utilization of EVs-based biomarkers, ultimately enhancing liver disease diagnosis, prognosis, and personalized treatment strategies.

Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.

Swelling-induced upregulation of miR-141-3p inhibits hepatocyte proliferation.

Background & Aims: MicroRNAs (miRNAs) act as a regulatory mechanism on a post-transcriptional level by repressing gene transcription/translation and play a central role in the cellular stress response. Osmotic changes occur in a variety of diseases including liver cirrhosis and hepatic encephalopathy. Changes in cell hydration and alterations of the cellular volume are major regulators of cell function and gene expression. In this study, the modulation of hepatic gene expression in response to hypoosmolarity was studied. Methods: mRNA analyses of normo- and hypoosmotic perfused rat livers by gene expression arrays were used to identify miRNA and their potential target genes associated with cell swelling preceding cell proliferation. Selected miR-141-3p was also investigated in isolated hepatocytes treated with miRNA mimic, cell stretching, and after partial hepatectomy. Inhibitor perfusion studies were performed to unravel signalling pathways responsible for miRNA upregulation. Results: Using genome-wide transcriptomic analysis, it was shown that hypoosmotic exposure led to differential gene expression in perfused rat liver. Moreover, miR-141-3p was upregulated by hypoosmolarity in perfused rat liver and in primary hepatocytes. In concert with this, miR-141-3p upregulation was prevented after Src-, Erk-, and p38-MAPK inhibition. Furthermore, luciferase reporter assays demonstrated that miR-141-3p targets cyclin dependent kinase 8 (Cdk8) mRNA. Partial hepatectomy transiently upregulated miR-141-3p levels just after the initiation of hepatocyte proliferation, whereas Cdk8 mRNA was downregulated. The mechanical stretching of rat hepatocytes resulted in miR-141-3p upregulation, whereas Cdk8 mRNA tended to decrease. Notably, the overexpression of miR-141-3p inhibited the proliferation of Huh7 cells. Conclusions: Src-mediated upregulation of miR-141-3p was found in hepatocytes in response to hypoosmotic swelling and mechanical stretching. Because of its antiproliferative function, miR-141-3p may counter-regulate the proliferative effects triggered by these stimuli. Lay summary: In this study, we identified microRNA 141-3p as an osmosensitive miRNA, which inhibits proliferation during liver cell swelling. Upregulation of microRNA 141-3p, controlled by Src-, Erk-, and p38-MAPK signalling, results in decreased mRNA levels of various genes involved in metabolic processes, macromolecular biosynthesis, and cell cycle progression.

Physical Interaction between Embryonic Stem Cell-Expressed Ras (ERas) and Arginase-1 in Quiescent Hepatic Stellate Cells.

Embryonic stem cell-expressed Ras (ERas) is an atypical constitutively active member of the Ras family and controls distinct signaling pathways, which are critical, for instance, for the maintenance of quiescent hepatic stellate cells (HSCs). Unlike classical Ras paralogs, ERas has a unique N-terminal extension (Nex) with as yet unknown function. In this study, we employed affinity pull-down and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and identified 76 novel binding proteins for human and rat ERas Nex peptides, localized in different subcellular compartments and involved in various cellular processes. One of the identified Nex-binding proteins is the nonmitochondrial, cytosolic arginase 1 (ARG1), a key enzyme of the urea cycle and involved in the de novo synthesis of polyamines, such as spermidine and spermine. Here, we show, for the first time, a high-affinity interaction between ERas Nex and purified ARG1 as well as their subcellular colocalization. The inhibition of ARG1 activity strikingly accelerates the activation of HSCs ex vivo, suggesting a central role of ARG1 activity in the maintenance of HSC quiescence.

Hepatic stellate cells: current state and open questions.

This review article summarizes 20 years of our research on hepatic stellate cells within the framework of two collaborative research centers CRC575 and CRC974 at the Heinrich Heine University. Over this period, stellate cells were identified for the first time as mesenchymal stem cells of the liver, and important functions of these cells in the context of liver regeneration were discovered. Furthermore, it was determined that the space of Disse - bounded by the sinusoidal endothelium and hepatocytes - functions as a stem cell niche for stellate cells. Essential elements of this niche that control the maintenance of hepatic stellate cells have been identified alongside their impairment with age. This article aims to highlight previous studies on stellate cells and critically examine and identify open questions and future research directions.

Improved Recovery from Liver Fibrosis by Crenolanib.

Chronic liver diseases are associated with excessive deposition of extracellular matrix proteins. This so-called fibrosis can progress to cirrhosis and impair vital functions of the liver. We examined whether the receptor tyrosine kinase (RTK) class III inhibitor Crenolanib affects the behavior of hepatic stellate cells (HSC) involved in fibrogenesis. Rats were treated with thioacetamide (TAA) for 18 weeks to trigger fibrosis. After TAA treatment, the animals received Crenolanib for two weeks, which significantly improved recovery from liver fibrosis. Because Crenolanib predominantly inhibits the RTK platelet-derived growth factor receptor-ß, impaired HSC proliferation might be responsible for this beneficial effect. Interestingly, blocking of RTK signaling by Crenolanib not only hindered HSC proliferation but also triggered their specification into hepatic endoderm. Endodermal specification was mediated by p38 mitogen-activated kinase (p38 MAPK) and c-Jun-activated kinase (JNK) signaling; however, this process remained incomplete, and the HSC accumulated lipids. JNK activation was induced by stress response-associated inositol-requiring enzyme-1α (IRE1α) in response to Crenolanib treatment, whereas ß-catenin-dependent WNT signaling was able to counteract this process. In conclusion, the Crenolanib-mediated inhibition of RTK impeded HSC proliferation and triggered stress responses, initiating developmental processes in HSC that might have contributed to improved recovery from liver fibrosis in TAA-treated rats.

Impaired integrin α5 /ß1 -mediated hepatocyte growth factor release by stellate cells of the aged liver.

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α5 /ß1 is an important mechanosensor in HSC involved in shear stress-induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and ß1 decreases in liver and HSC from aged rats. CRISPR/Cas9-mediated integrin α5 and ß1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin ß1 remains unaffected. In the aged liver, laminin ß2 and γ1 protein chains as components of laminin-521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin-521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence-associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α5 /ß1 in HSC contributes to age-related reduction of ECM and HGF release that could affect liver regeneration.

iRhom2 inhibits bile duct obstruction-induced liver fibrosis.

Chronic liver disease can induce prolonged activation of hepatic stellate cells, which may result in liver fibrosis. Inactive rhomboid protein 2 (iRhom2) is required for the maturation of A disintegrin and metalloprotease 17 (ADAM17, also called TACE), which is responsible for the cleavage of membrane-bound tumor necrosis factor-α (TNF-α) and its receptors (TNFRs). Here, using the murine bile duct ligation (BDL) model, we showed that the abundance of iRhom2 and activation of ADAM17 increased during liver fibrosis. Consistent with this, concentrations of ADAM17 substrates were increased in plasma samples from mice after BDL and in patients suffering from liver cirrhosis. We observed increased liver fibrosis, accelerated disease progression, and an increase in activated stellate cells after BDL in mice lacking iRhom2 (Rhbdf2-/- ) compared to that in controls. In vitro primary mouse hepatic stellate cells exhibited iRhom2-dependent shedding of the ADAM17 substrates TNFR1 and TNFR2. In vivo TNFR shedding after BDL also depended on iRhom2. Treatment of Rhbdf2-/- mice with the TNF-α inhibitor etanercept reduced the presence of activated stellate cells and alleviated liver fibrosis after BDL. Together, these data suggest that iRhom2-mediated inhibition of TNFR signaling protects against liver fibrosis.

Space of Disse: a stem cell niche in the liver.

Recent evidence indicates that the plasticity of preexisting hepatocytes and bile duct cells is responsible for the appearance of intermediate progenitor cells capable of restoring liver mass after injury without the need of a stem cell compartment. However, mesenchymal stem cells (MSCs) exist in all organs and are associated with blood vessels which represent their perivascular stem cell niche. MSCs are multipotent and can differentiate into several cell types and are known to support regenerative processes by the release of immunomodulatory and trophic factors. In the liver, the space of Disse constitutes a stem cell niche that harbors stellate cells as liver resident MSCs. This perivascular niche is created by extracellular matrix proteins, sinusoidal endothelial cells, liver parenchymal cells and sympathetic nerve endings and establishes a microenvironment that is suitable to maintain stellate cells and to control their fate. The stem cell niche integrity is important for the behavior of stellate cells in the normal, regenerative, aged and diseased liver. The niche character of the space of Disse may further explain why the liver can become an organ of extra-medullar hematopoiesis and why this organ is frequently prone to tumor metastasis.

IL-6 Trans-signaling Controls Liver Regeneration After Partial Hepatectomy.

Interleukin-6 (IL-6) is critically involved in liver regeneration after partial hepatectomy (PHX). Previous reports suggest that IL-6 trans-signaling through the soluble IL-6/IL-6R complex is involved in this process. However, the long-term contribution of IL-6 trans-signaling for liver regeneration after PHX is unknown. PHX-induced generation of the soluble IL-6R by ADAM (a disintegrin and metallo) proteases enables IL-6 trans-signaling, in which IL-6 forms an agonistic complex with the soluble IL-6 receptor (sIL-6R) to activate all cells expressing the signal-transducing receptor chain glycoprotein 130 (gp130). In contrast, without activation of ADAM proteases, IL-6 in complex with membrane-bound IL-6R and gp130 activates classic signaling. Here, we describe the generation of IL-6 trans-signaling mice, which exhibit boosted IL-6 trans-signaling and abrogated classic signaling by genetic conversion of all membrane-bound IL-6R into sIL-6R proteins phenocopying hyperactivation of ADAM-mediated shedding of IL-6R as single substrate. Importantly, although IL-6R deficient mice were strongly affected by PHX, survival and regeneration of IL-6 trans-signaling mice was indistinguishable from control mice, demonstrating that IL-6 trans-signaling fully compensates for disabled classic signaling in liver regeneration after PHX. Moreover, we monitored the long-term consequences of global IL-6 signaling inhibition versus IL-6 trans-signaling selective blockade after PHX by IL-6 monoclonal antibodies and soluble glycoprotein 130 as fragment crystallizable fusion, respectively. Both global IL-6 blockade and selective inhibition of IL-6 trans-signaling results in a strong decrease of overall survival after PHX, accompanied by decreased signal transducer and activator of transcription 3 phosphorylation and proliferation of hepatocytes. Mechanistically, IL-6 trans-signaling induces hepatocyte growth factor production by hepatic stellate cells. Conclusion: IL-6 trans-signaling, but not classic signaling, controls liver regeneration following PHX.

Transplanted Human Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Support Liver Regeneration in Gunn Rats.

Gunn rats bear a mutation within the uridine diphosphate glucuronosyltransferase-1a1 (Ugt1a1) gene resulting in high serum bilirubin levels as seen in Crigler-Najjar syndrome. In this study, the Gunn rat was used as an animal model for heritable liver dysfunction. Induced mesenchymal stem cells (iMSCs) derived from embryonic stem cells (H1) and induced pluripotent stem cells were transplanted into Gunn rats after partial hepatectomy. The iMSCs engrafted and survived in the liver for up to 2 months. The transplanted iMSCs differentiated into functional hepatocytes as evidenced by partially suppressed hyperbilirubinemia and expression of multiple human-specific hepatocyte markers such as albumin, hepatocyte nuclear factor 4α, UGT1A1, cytokeratin 18, bile salt export pump, multidrug resistance protein 2, Na/taurocholate-cotransporting polypeptide, and α-fetoprotein. These findings imply that transplanted human iMSCs can contribute to liver regeneration in vivo and thus represent a promising tool for the treatment of inherited liver diseases.

Laminin-521 promotes quiescence in isolated stellate cells from rat liver.

The laminin α5 protein chain is an element of basement membranes and important to maintain stem cells. Hepatic stellate cells (HSC) are liver-resident mesenchymal stem cells, which reside in a quiescent state on a basement membrane-like structure in the space of Dissé. In the present study, laminin α5 chain was detected in the space of Dissé of normal rat liver. Since HSC are critical for liver regeneration and can contribute to fibrosis in chronic liver diseases, the effect of laminins on HSC maintenance was investigated. Therefore, isolated rat HSC were seeded on uncoated polystyrene (PS) or PS coated with either laminin-521 (PS/LN-521) or laminin-211 (PS/LN-211). PS/LN-521 improved HSC adhesion and better preserved their retinoid stores as well as quiescence- and stem cell-associated phenotype, whereas HSC on PS/LN-211 or PS developed into myofibroblasts-like cells. To improve the homogeneity as well as the presentation of laminin molecules on the culture surface to HSC, laminin-functionalized, gold-nanostructured glass surfaces were generated. This approach further enhanced the expression of quiescence-associated genes in HSC. In conclusion, the results indicate that LN-521 supports the quiescent state of HSC and laminin α5 can be regarded as an important element of their niche in the space of Dissé.

Combined Methylome and Transcriptome Analysis During Rat Hepatic Stellate Cell Activation.

Hepatic stellate cells (HSCs) are mesenchymal stem cells (MSCs) of the liver. They are unique among MSCs, since HSCs remain in a quiescent, retinoid-storing state in the normal liver but become activated after liver injury and contribute to tissue repair. The epigenetic mechanisms accompanying the transition of HSCs from a quiescent to an activated state are in the focus of the present study. We investigated the methylome and transcriptome during this process and observed profound changes. While the promoter methylation correlated negatively with gene expression, the gene-body methylation revealed no clear correlation. Most genes with altered expression were associated with cell differentiation. Among them, Wilms tumor 1 (Wt1) and Deltex4 (Dtx4) genes were identified as epigenetically regulated. Since HSCs were reported to derive from multipotent Wt1-positive cells and many differentially expressed genes were associated with cell differentiation during their activation, epigenetic alterations are presumably required to enable HSC development.

Mechanisms of Tauroursodeoxycholate-Mediated Hepatoprotection.

Ursodeoxycholate and its taurine conjugate tauroursodeoxycholate (TUDC) promote choleresis by triggering the insertion of transport proteins for bile acids into the canalicular and basolateral membranes of hepatocytes. In addition, TUDC exerts hepatoprotective and anti-apoptotic effects, can counteract the action of toxic bile acids and reduce endoplasmic reticulum stress. TUDC can also initiate the differentiation of multipotent mesenchymal stem cells (MSC) including hepatic stellate cells and promote their development into hepatocyte-like cells. Although the hepatoprotective and choleretic action of TUDC is empirically used in clinical medicine since decades, the underlying molecular mechanisms remained largely unclear. Since TUDC has little or no potency to activate known bile acid receptors, such as farnesoid X receptor and transmembrane G protein-coupled bile acid receptor, other receptors must be involved in TUDC-mediated signaling. Recent research demonstrates that integrins serve as sensors for TUDC. After binding of TUDC to α5ß1-integrin, the ß1-integrin subunit becomes activated through a conformational change, thereby triggering integrin signaling with the downstream activation of focal adhesion kinase, c-Src, the epidermal growth factor receptor and activation of the mitogen-activated protein kinases, Erks and p38. These events trigger choleresis through a coordinated insertion of the sodium-taurocholate cotransporting polypeptide into the basolateral membrane and of the bile salt export pump into the canalicular membrane. In addition to its choleretic action, TUDC-induced integrin activation triggers a cyclic adenosine monophosphate-dependent protein kinase A activation in hepatocytes, which provides the basis for the anti-apoptotic effect of TUDC. On the other hand, the TUDC-induced stimulation of MSC differentiation appears not to be mediated by integrins. This article gives a brief overview about our work on the signaling network-mediating hepatoprotection by TUDC.

Isolation and characterization of vesicular and non-vesicular microRNAs circulating in sera of partially hepatectomized rats.

Circulating microRNAs are protected from degradation by their association with either vesicles or components of the RNAi machinery. Although increasing evidence indicates that cell-free microRNAs are transported in body fluids by different types of vesicles, current research mainly focuses on the characterization of exosome-associated microRNAs. However, as isolation and characterization of exosomes is challenging, it is yet unclear whether exosomes or other vesicular elements circulating in serum are the most reliable source for discovering disease-associated biomarkers. In this study, circulating microRNAs associated to the vesicular and non-vesicular fraction of sera isolated from partially hepatectomized rats were measured. Here we show that independently from their origin, levels of miR-122, miR-192, miR-194 and Let-7a are up-regulated two days after partial hepatectomy. The inflammation-associated miR-150 and miR-155 are up-regulated in the vesicular-fraction only, while the regeneration-associated miR-21 and miR-33 are up-regulated in the vesicular- and down-regulated in the non-vesicular fraction. Our study shows for the first time the modulation of non-vesicular microRNAs in animals recovering from partial hepatectomy, suggesting that, in the search for novel disease-associated biomarkers, the profiling of either vesicular or non-vesicular microRNAs may be more relevant than the analysis of microRNAs isolated from unfractionated serum.

The Role of Embryonic Stem Cell-expressed RAS (ERAS) in the Maintenance of Quiescent Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) were recently identified as liver-resident mesenchymal stem cells. HSCs are activated after liver injury and involved in pivotal processes, such as liver development, immunoregulation, regeneration, and also fibrogenesis. To date, several studies have reported candidate pathways that regulate the plasticity of HSCs during physiological and pathophysiological processes. Here we analyzed the expression changes and activity of the RAS family GTPases and thereby investigated the signaling networks of quiescent HSCs versus activated HSCs. For the first time, we report that embryonic stem cell-expressed RAS (ERAS) is specifically expressed in quiescent HSCs and down-regulated during HSC activation via promoter DNA methylation. Notably, in quiescent HSCs, the high level of ERAS protein correlates with the activation of AKT, STAT3, mTORC2, and HIPPO signaling pathways and inactivation of FOXO1 and YAP. Our data strongly indicate that in quiescent HSCs, ERAS targets AKT via two distinct pathways driven by PI3Kα/δ and mTORC2, whereas in activated HSCs, RAS signaling shifts to RAF-MEK-ERK. Thus, in contrast to the reported role of ERAS in tumor cells associated with cell proliferation, our findings indicate that ERAS is important to maintain quiescence in HSCs.

Bile acids induce hepatic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSC) have the potential to differentiate into multiple cell lineages and their therapeutic potential has become obvious. In the liver, MSC are represented by stellate cells which have the potential to differentiate into hepatocytes after stimulation with growth factors. Since bile acids can promote liver regeneration, their influence on liver-resident and bone marrow-derived MSC was investigated. Physiological concentrations of bile acids such as tauroursodeoxycholic acid were able to initiate hepatic differentiation of MSC via the farnesoid X receptor and transmembrane G-protein-coupled bile acid receptor 5 as investigated with knockout mice. Notch, hedgehog, transforming growth factor-ß/bone morphogenic protein family and non-canonical Wnt signalling were also essential for bile acid-mediated differentiation, whereas ß-catenin-dependent Wnt signalling was able to attenuate this process. Our findings reveal bile acid-mediated signalling as an alternative way to induce hepatic differentiaion of stem cells and highlight bile acids as important signalling molecules during liver regeneration.

The Influence of Oxygen on the Proliferative Capacity and Differentiation Potential of Lacrimal Gland-Derived Mesenchymal Stem Cells.

PURPOSE: The application of lacrimal gland-derived mesenchymal stem cells (LG-MSC) for the regeneration of lacrimal gland tissue could result in a novel therapy for dry-eye syndrome. To optimize the culture conditions, the purpose of this study was to evaluate the influence of low oxygen on phenotype, differentiation potential, proliferative, and regenerative capacity of murine LG-MSC. METHODS: Murine LG-MSC were cultured in 21% and 5% oxygen and characterized by flow cytometry, cell sorter assisted proliferation-, and colony forming unit-assays. Reactive oxygen species (ROS) levels as well as lineage differentiation were evaluated. The effect of conditioned medium of LG-MSC from both oxygen conditions (CM MSC 21%, respectively, CM MSC 5%) on lacrimal gland epithelial cells (LG-EC) was examined in wound healing and proliferation assays. RESULTS: Cells under both culture conditions revealed differentiation potential and presented a MSC-specific flow cytometric phenotype. In 5% oxygen, cells yielded less ROS, showed a stable morphology, higher colony forming potential, and an increased proliferation capacity. Five percent oxygen significantly increased the number of CD44+ LG-MSC. Furthermore, CM MSC 5% significantly enhanced migration and proliferation in LG-EC. CONCLUSIONS: In vitro expansion in low oxygen preserves the proliferation capacity and differentiation potential of LG-MSC and increases the effects of conditioned medium on migration and proliferation in LG-EC. Therefore, expansion in low oxygen seems to be an excellent method, to obtain vital MSC. Also, an increased number of LG-MSC expressing CD44 was observed under low oxygen, which might be a valuable marker to identify a potent MSC subpopulation.

Bile acids and stellate cells.

Hepatic stellate cells are mainly known for their contribution to fibrogenesis in chronic liver diseases, but their identity and function in normal liver remain unclear. They were recently identified as liver-resident mesenchymal stem cells (MSCs), which can differentiate not only into adipocytes and osteocytes, but also into liver epithelial cells such as hepatocytes and bile duct cells as investigated in vitro and in vivo. During hepatic differentiation, stellate cells and other MSCs transiently develop into liver progenitor cells with epithelial characteristics before hepatocytes are established. Transplanted stellate cells from the liver and pancreas are able to contribute to liver regeneration in stem cell-based liver injury models and can also home into the bone marrow, which is in line with their classification as MSCs. There is experimental evidence that bile acids support liver regeneration and are able to activate signaling pathways in hepatic stellate cells. For this reason, it is important to analyze the influence of bile acids on developmental fate decisions of hepatic stellate cells and other MSC populations.