Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.

(-)-Epigallocatechin-3-gallate induced apoptosis by dissociation of c-FLIP/Ku70 complex in gastric cancer cells.

Anti-cancer properties of (-)-epigallocatechin-3-gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE-inhibitory protein (c-FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti-cancer role of EGCG in gastric cancer (GC) cells through dissociation of c-FLIP/Ku70 complex. MKN-45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT-PCR. Protein expression of c-FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c-FLIP/Ku70 complex as well as Ku70 translocation were studied by sub-cellular fractionation and co-immunoprecipitation. EGCG induced apoptosis in MKN-45 cells with substantial up-regulation of P53 and P21, down-regulation of c-Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c-FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c-FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.

Intercellular communication among liver cells in the perisinusoidal space of the injured liver: Pathophysiology and therapeutic directions.

Hepatic stellate cells (HSCs) in the perisinusoidal space are surrounded by hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and other resident immune cells. In the normal liver, HSCs communicate with these cells to maintain normal liver functions. However, after chronic liver injury, injured hepatocytes release several proinflammatory mediators, reactive oxygen species, and damage-associated molecular patterns into the perisinusoidal space. Consequently, such alteration activates quiescent HSCs to acquire a myofibroblast-like phenotype and express high amounts of transforming growth factor-ß1, angiopoietins, vascular endothelial growth factors, interleukins 6 and 8, fibril forming collagens, laminin, and E-cadherin. These phenotypic and functional transdifferentiation lead to hepatic fibrosis with a typical abnormal extracellular matrix synthesis and disorganization of the perisinusoidal space of the injured liver. Those changes provide a favorable environment that regulates tumor cell proliferation, migration, adhesion, and survival in the perisinusoidal space. Such tumor cells by releasing transforming growth factor-ß1 and other cytokines, will, in turn, activate and deeply interact with HSCs via a bidirectional loop. Furthermore, hepatocellular carcinoma-derived mediators convert HSCs and macrophages into protumorigenic cell populations. Thus, the perisinusoidal space serves as a critical hub for activating HSCs and their interactions with other cell types, which cause a variety of liver diseases such as hepatic inflammation, fibrosis, cirrhosis, and their complications, such as portal hypertension and hepatocellular carcinoma. Therefore, targeting the crosstalk between activated HSCs and tumor cells/immune cells in the tumor microenvironment may also support a promising therapeutic strategy.

Bioscreening and pre-clinical evaluation of the impact of bioactive molecules from Ptychotis verticillata on the multilineage potential of mesenchymal stromal cells towards immune- and inflammation-mediated diseases.

OBJECTIVE AND DESIGN: Mesenchymal stromal cells (MSCs) are currently used in cell reparative medicine due to their trophic and ant-inflammatory properties. The modulation of stem cell properties by phytochemicals has been suggested as a tool to empower their tissue repair capacity. In vitro, MSCs are characterized by their tri-lineage potential that holds great interest for tissue regeneration. Ptychotis Verticillata (PV), an aromatic and medicinal plant, may be thus used to modulate the in vitro multilineage potential of MSCs. MATERIALS AND METHODS: We screened the impact of PV-derived essential oil and their bioactive molecules (thymol and carvacrol) on the in vitro multilineage potential of MSCs. Different concentrations and incubation times of these compounds were assessed during the osteogenesis and adipogenesis of MSCs. RESULTS: The analysis of 75 conditions indicates that these compounds are biologically active by promoting two major differentiation lineages from MSCs. In a time- and dose-dependent manner, thymol and carvacrol increased the osteogenesis and adipogenesis. CONCLUSION: According to these preliminary observations, the addition of PV extract may stimulate the tissue regenerative and repair functions of MSCs. Further optimization of compound extraction and characterization from PV as well as cell treatment conditions should increase their therapeutic value in combination with MSCs.

Advanced therapeutic modalities in hepatocellular carcinoma: Novel insights.

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is usually a latent and asymptomatic malignancy caused by different aetiologies, which is a result of various aberrant molecular heterogeneity and often diagnosed at advanced stages. The incidence and prevalence have significantly increased because of sedentary lifestyle, diabetes, chronic infection with hepatotropic viruses and exposure to aflatoxins. Due to advanced intra- or extrahepatic metastasis, recurrence is very common even after radical resection. In this paper, we highlighted novel therapeutic modalities, such as molecular-targeted therapies, targeted radionuclide therapies and epigenetic modification-based therapies. These topics are trending headlines and their combination with cell-based immunotherapies, and gene therapy has provided promising prospects for the future of HCC treatment. Moreover, a comprehensive overview of current and advanced therapeutic approaches is discussed and the advantages and limitations of each strategy are described. Finally, very recent and approved novel combined therapies and their promising results in HCC treatment have been introduced.

Immuno-comparative screening of adult-derived human liver stem/progenitor cells for immune-inflammatory-associated molecules.

OBJECTIVE: One of the main challenges in liver cell therapy is the replacement of damaged cells and the induction of a tolerogenic microenvironment to promote graft acceptance by the recipient. Adult-derived human liver stem/progenitor cells (ADHLSCs) are currently evaluated at the clinical levels as a promising pro-regenerative and immune-modulatory tool. The expression profile of several immunological molecules may influence the local immune-inflammatory response and, therefore, modulate the tissue healing process. To increase the quality and safety of ADHLSCs before transplantation requires an appropriate analysis and characterization of their pattern expression of immune-inflammatory-associated molecules. METHODS: The expression of 27 molecules belonging to T-cell co-stimulatory pathway, CD47 partners, Ikaros family, CD300 family and TNF family were analyzed using flow cytometry. We compared their expression profiles to PBMCs, hepatocytes and ADHLSCs in both expansion and after hepatogenic differentiation culture conditions. RESULTS: This original immuno-comparative screening revealed that liver cell populations do not constitutively present significant immunological pattern compared to PBMCs. Moreover, our findings highlight that neither the expansion nor the hepatogenic differentiation induces the expression of immune-inflammatory molecules. The detailed expression characteristics (percentage of positive cells and median fluorescence intensity) of each molecule were analyzed and presented. CONCLUSION: By analyzing 27 relevant molecules, our immuno-comparative screening demonstrates that ADHLSCs keep a non-immunogenic profile independent of their expansion or hepatogenic differentiation state. Accordingly, the immunological profile of ADHLSCs seems to support their safe and efficient use in liver tissue therapeutic repair strategy.

New Anti-Leukemic Effect of Carvacrol and Thymol Combination through Synergistic Induction of Different Cell Death Pathways.

Acute myeloid leukemia (AML) is a cancer of the myeloid lineage of blood cells, and treatment for AML is lengthy and can be very expensive. Medicinal plants and their bioactive molecules are potential candidates for improving human health. In this work, we studied the effect of Ptychotis verticillata (PV) essential oil and its derivatives, carvacrol and thymol, in AML cell lines. We demonstrated that a combination of carvacrol and thymol induced tumor cell death with low toxicity on normal cells. Mechanistically, we highlighted that different molecular pathways, including apoptosis, oxidative, reticular stress, autophagy, and necrosis, are implicated in this potential synergistic effect. Using quantitative RT-PCR, Western blotting, and apoptosis inhibitors, we showed that cell death induced by the carvacrol and thymol combination is caspase-dependent in the HL60 cell line and caspase-independent in the other cell lines tested. Further investigations should focus on improving the manufacturing of these compounds and understanding their anti-tumoral mechanisms of action. These efforts will lead to an increase in the efficiency of the oncotherapy strategy regarding AML.

Accurate and live peroxisome biogenesis evaluation achieved by lentiviral expression of a green fluorescent protein fused to a peroxisome targeting signal 1.

Peroxisomes are ubiquitous organelles formed by peroxisome biogenesis (PB). During PB, peroxisomal matrix proteins harboring a peroxisome targeting signal (PTS) are imported inside peroxisomes by peroxins, encoded by PEX genes. Genetic alterations in PEX genes lead to a spectrum of incurable diseases called Zellweger spectrum disorders (ZSD). In vitro drug screening is part of the quest for a cure in ZSD by restoring PB in ZSD cell models. In vitro PB evaluation is commonly achieved by immunofluorescent staining or transient peroxisome fluorescent reporter expression. Both techniques have several drawbacks (cost, time-consuming technique, etc.) which we overcame by developing a third-generation lentiviral transfer plasmid expressing an enhanced green fluorescent protein fused to PTS1 (eGFP-PTS1). By eGFP-PTS1 lentiviral transduction, we quantified PB and peroxisome motility in ZSD and control mouse and human fibroblasts. We confirmed the stable eGFP-PTS1 expression along cell passages. eGFP signal analysis distinguished ZSD from control eGFP-PTS1-transduced cells. Live eGFP-PTS1 transduced cells imaging quantified peroxisomes motility. In conclusion, we developed a lentiviral transfer plasmid allowing stable eGFP-PTS1 expression to study PB (deposited on Addgene: #133282). This tool meets the needs for in vitro PB evaluation and ZSD drug discovery.

ß-radiating radionuclides in cancer treatment, novel insight into promising approach.

Targeted radionuclide therapy, known as molecular radiotherapy is a novel therapeutic module in cancer medicine. ß-radiating radionuclides have definite impact on target cells via interference in cell cycle and particular signalings that can lead to tumor regression with minimal off-target effects on the surrounding tissues. Radionuclides play a remarkable role not only in apoptosis induction and cell cycle arrest, but also in the amelioration of other characteristics of cancer cells. Recently, application of novel ß-radiating radionuclides in cancer therapy has been emerged as a promising therapeutic modality. Several investigations are ongoing to understand the underlying molecular mechanisms of ß-radiating elements in cancer medicine. Based on the radiation dose, exposure time and type of the ß-radiating element, different results could be achieved in cancer cells. It has been shown that ß-radiating radioisotopes block cancer cell proliferation by inducing apoptosis and cell cycle arrest. However, physical characteristics of the ß-radiating element (half-life, tissue penetration range, and maximum energy) and treatment protocol determine whether tumor cells undergo cell cycle arrest, apoptosis or both and to which extent. In this review, we highlighted novel therapeutic effects of ß-radiating radionuclides on cancer cells, particularly apoptosis induction and cell cycle arrest.

Bisphenol A exposure disrupts aspartate transport in HepG2 cells.

The liver is the organ responsible for bisphenol A (BPA) metabolism, an environmental chemical agent. Exposure to this toxin is associated with liver abnormalities and dysfunction. An important role played by excitatory amino acid transporters (EAATs) of the slc1 gene family has been reported in liver injuries. To gain insight into a plausible effect of BPA exposure in the liver glutamate/aspartate transport, using the human hepatoblastoma cell line HepG2, we report a BPA-dependent dynamic regulation of SLC1A3 and SLC1A2. Through the use of radioactive [3 H]- d-aspartate uptake experiments and immunochemical approaches, we characterized time and dose-dependent regulation of the protein levels and function of these transporters after acute exposure to BPA. An increase in nuclear Yin Yang 1 was found. These results suggest an important involvement of the EAATs in liver physiology and its disruption after acute BPA exposure.

The Transcription Factor 7-Like 2-Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1 Alpha Axis Connects Mitochondrial Biogenesis and Metabolic Shift with Stem Cell Commitment to Hepatic Differentiation.

Increasing evidence supports that modifications in the mitochondrial content, oxidative phosphorylation (OXPHOS) activity, and cell metabolism influence the fate of stem cells. However, the regulators involved in the crosstalk between mitochondria and stem cell fate remains poorly characterized. Here, we identified a transcriptional regulatory axis, composed of transcription factor 7-like 2 (TCF7L2) (a downstream effector of the Wnt/ß-catenin pathway, repressed during differentiation) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) (the master regulator of mitochondrial biogenesis, induced during differentiation), coupling the loss of pluripotency and early commitment to differentiation, to the initiation of mitochondrial biogenesis and metabolic shift toward OXPHOS. PGC-1α induction during differentiation is required for both mitochondrial biogenesis and commitment to the hepatocytic lineage, and TCF7L2 repression is sufficient to increase PGC-1α expression, mitochondrial biogenesis and OXPHOS activity. We further demonstrate that OXPHOS activity is required for the differentiation toward the hepatocytic lineage, thus providing evidence that bi-directional interactions control stem cell differentiation and mitochondrial abundance and activity. Stem Cells 2017;35:2184-2197.

Hepatic fibrosis: It is time to go with hepatic stellate cell-specific therapeutic targets.

Hepatic fibrosis is a pathological lesion, characterized by the progressive accumulation of extracellular matrix (ECM) in the perisinusoidal space and it is a major problem in chronic liver diseases. Phenotypic activation of hepatic stellate cells (HSC) plays a central role in the progression of hepatic fibrosis. Retardation of proliferation and clearance of activated HSCs from the injured liver is an appropriate therapeutic strategy for the resolution and treatment of hepatic fibrosis. Clearance of activated HSCs from the injured liver by autophagy inhibitors, proapoptotic agents and senescence inducers with the high affinity toward the activated HSCs may be the novel therapeutic strategy for the treatment of hepatic fibrosis in the near future.

Human hepatic stellate cells and inflammation: A regulated cytokine network balance.

AIM: Uncertainty about the safety of cell therapy continues to be a major challenge to the medical community. Inflammation and the associated immune response represent a major safety concern hampering the development of long-term clinical therapy. In vivo interactions between the cell graft and the host immune system are mediated by functional environmental sensors and stressors that play significant roles in the immunobiology of the graft. Within this context, human liver stellate cells (HSC) demonstrated marked immunological plasticity that has main importance for future liver cell therapy application. METHODS: By using qPCR technique, we established the cytokine gene expression profile of HSCs and investigated the effect of an inflammatory environment on the immunobiology of HSCs. RESULTS AND DISCUSSION: HSCs present a specific immunological profile as demonstrated by the expression and modulation of major immunological cytokines. Under constitutive conditions, the cytokine pattern expressed by HSCs was characterized by the high expression of IL-6. Inflammation critically modulated the expression of major immunological cytokines. As evidenced by the induction of the expression of several inflammatory genes, HSCs acquire a pro-inflammatory profile that ultimately might have critical implications for their immunological shape. CONCLUSION: These new observations have to be taken into account in any future liver cell therapy application based on the use of HSCs.

Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression.

BACKGROUND: Proliferation of hepatic stellate cells (HSCs) plays a pivotal role in the progression of liver fibrosis consequent to chronic liver injury. Silibinin, a flavonoid compound, has been shown to possess anti-fibrogenic effects in animal models of liver fibrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fibrogenic effect. METHODS: The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin (10, 50 and 100 µmol/L) for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay. RESULTS: Silibinin inhibits LX-2 cell proliferation in dose- and time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regulation was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinin-inhibited proliferation of LX-2 cells. CONCLUSION: The anti-proliferative effect of silibinin on LX-2 human stellate cells is via the inhibition of the expressions of various cell cycle targets including p27, Akt and sirtuin signaling.

Influence of inflammation on the immunological profile of adult-derived human liver mesenchymal stromal cells and stellate cells.

BACKGROUND AIMS: Stem cell therapy for liver diseases has recently emerged as a promising alternative to liver transplantation. Eligible cells should have an appropriate immunophenotype. The aim of the present study was to define the immunological profile of two human liver-derived mesenchymal stromal cell populations, namely, stem cells (ADHLSC) and hepatic stellate cells (HSC). METHODS: The study was conducted under normal and inflammatory conditions with the use of human bone marrow mesenchymal stromal cells (BM-MSC) as reference. RESULTS: Like BM-MSC and ADHLSC, HSC were negative for hematopoietic (CD45) and endothelial (CD34) markers but positive for stromal markers. All cell types were constitutively positive for HLA class I and negative for human leukocyte antigen (HLA) class II and co-stimulatory molecules (CD80, CD86, CD134 and CD252). Inflammation induced the expression of CD40 in all cell types, but the highest values were observed on HSCs; high CD252 expression was only observed on HSC as compared with ADHLSC and BM-MSC. The expression of various adhesion molecules (CD54, CD58, CD106 and CD166) was dissimilar in these three cell types and was differentially influenced by inflammation as well. ADHLSC and HSC constitutively expressed the immunosuppressive molecule HLA-G, whereas CD274 expression was induced by inflammation, as in the case of BM-MSC. Moreover, all cell types expressed the two major natural killer ligands CD112 and CD115. CONCLUSIONS: Toll-like receptors (TLR) 1, 3, 4 and 6 messenger RNA was expressed by both cell types, whereas TLR 2, 5, 7, 9 and 10 were only expressed by ADHLSC. Inflammation increased the expression of TLR 2 and 3 by ADHLSC and HSC. Finally, both liver-derived cell types were immunosuppressive because they inhibited the proliferation of mitogen-activated T cells.

Glutamate-dependent translational control through ribosomal protein S6 phosphorylation in cultured bergmann glial cells.

Glutamate (Glu) the main excitatory neurotransmitter of the central nervous system regulates gene expression at different levels through the activation of specific membrane receptors and transporters expressed in neurons and glia cells. A membrane to nucleus signaling cascade triggered by this neurotransmitter has been described in cultured cerebellar Bergmann glia cells isolated from chick embryos. Furthermore, it has also been described that Glu receptors activation is linked to a modulation of [(35)S]-methionine incorporation into newly synthesized polypeptides. In order to gain insight into the signal transduction cascades that participate in this effect, in the present study we characterized the phosphorylation of a critical component of the translational machinery, namely the ribosomal protein S6. The phosphorylation sites in rpS6 have been mapped to five clustered residues, Ser235, Ser236, Ser240, Ser244 and Ser247. Nevertheless, Ser236 phosphorylation is the primary phosphorylation site. The kinases responsible of this modification are p70(S6K) and p90(RSK). rpS6 phosphorylation increases the affinity of 40s subunit for mRNAs and thus facilitates translational initiation. Glutamate exposure of cultured cerebellar Bergmann glia cells results in a time- and dose-dependent increase in rpS6 phosphorylation. This effect is mainly observed at cytoplasm, and involves the phosphoinositol-3 kinase/protein kinase B pathway. Our results favor the notion of a continuous neuronal signaling to glia cells that regulates the proteome of these cells not only at the transcriptional level but also at the level of protein synthesis.

Differentiated umbilical cord matrix stem cells as a new in vitro model to study early events during hepatitis B virus infection.

UNLABELLED: The role of cell differentiation state on hepatitis B virus (HBV) replication has been well demonstrated, whereas how it determines cell susceptibility to HBV entry is far less understood. We previously showed that umbilical cord matrix stem cells (UCMSC) can be differentiated towards hepatocyte-like cells in vitro. In this study we infected undifferentiated (UD-) and differentiated (D-) UCMSCs with HBV and studied the infection kinetics, comparing them to primary human hepatocytes (PHHs). UD-UCMSCs, although permissive to viral binding, had a very limited uptake capacity, whereas D-UCMSCs showed binding and uptake capabilities similar to PHHs. Likewise, asialoglycoprotein receptor (ASGPR) was up-regulated in UCMSCs upon differentiation. In D-UCMSCs, a dose-dependent inhibition of HBV binding and uptake was observed when ASGPR was saturated with known specific ligands. Subsequent viral replication was shown in D-UCMSCs but not in UD-UCMSCs. Susceptibility of UCMSCs to viral replication correlated with the degree of differentiation. Replication efficiency was low compared to PHHs, but was confirmed by (1) a dose-dependent inhibition by specific antiviral treatment using tenofovir; (2) the increase of viral RNAs along time; (3) de novo synthesis of viral proteins; and (4) secretion of infectious viral progeny. CONCLUSION: UCMSCs become supportive of the entire HBV life cycle upon in vitro hepatic differentiation. Despite low replication efficiency, D-UCMSCs proved to be fully capable of HBV uptake. Overall, UCMSCs are a unique human, easily available, nontransformed, in vitro model of HBV infection that could prove useful to study early infection events and the role of the cell differentiation state on such events.

The immunogenic profile and immunomodulatory function of mesenchymal stromal / stem cells in the presence of Ptychotis verticillata.

Mesenchymal stromal/stem cells (MSCs) are considered to be a promising immunotherapeutic tool due to their easy accessibility, culture expansion possibilities, safety profile, and immunomodulatory properties. Although several studies have demonstrated the therapeutic effects of MSCs, their efficacy needs to be improved while also preserving their safety. It has been suggested that cell homeostasis may be particularly sensitive to plant extracts. The impact of natural compounds on immunity is thus a fascinating and growing field. Ptychotis verticillata and its bioactive molecules, carvacrol and thymol, are potential candidates for improving MSC therapeutic effects. They can be used as immunotherapeutic agents to regulate MSC functions and behavior during immunomodulation. Depending on their concentrations and incubation time, these compounds strengthened the immunomodulatory functions of MSCs while maintaining their immune-evasive profile. Incubating MSCs with carvacrol and thymol does not alter their hypoimmunogenicity, as no induction of the allogeneic immune response was observed. MSCs also showed enhanced abilities to reduce the proliferation of activated T cells. Thus, MSCs are immunologically responsive to bioactive molecules derived from PV. The bioactivity may depend on the whole phyto-complex of the oil. These findings may contribute to the development of safe and efficient immunotherapeutic MSCs by using medicinal plant-derived active molecules.

Immune regulation and therapeutic application of T regulatory cells in liver diseases.

CD4+ CD25+ FOXP3+ T regulatory cells (Tregs) are a subset of the immunomodulatory cell population that can inhibit both innate and adaptive immunity by various regulatory mechanisms. In hepatic microenvironment, proliferation, plasticity, migration, and function of Tregs are interrelated to the remaining immune cells and their secreted cytokines and chemokines. In normal conditions, Tregs protect the liver from inflammatory and auto-immune responses, while disruption of this crosstalk between Tregs and other immune cells may result in the progression of chronic liver diseases and the development of hepatic malignancy. In this review, we analyze the deviance of this protective nature of Tregs in response to chronic inflammation and its involvement in inducing liver fibrosis, cirrhosis, and hepatocellular carcinoma. We will also provide a detailed emphasis on the relevance of Tregs as an effective immunotherapeutic option for autoimmune diseases, liver transplantation, and chronic liver diseases including liver cancer.

Successful isolation of liver progenitor cells by aldehyde dehydrogenase activity in naïve mice.

UNLABELLED: The role of progenitor cells in liver repair and fibrosis has been extensively described, but their purification remains a challenge, hampering their characterization and use in regenerative medicine. To address this issue, we developed an easy and reproducible liver progenitor cell (LPC) isolation strategy based on aldehyde dehydrogenase (ALDH) activity, a common feature shared by many progenitor cells. We demonstrate that a subset of nonparenchymal mouse liver cells displays high levels of ALDH activity, allowing the isolation of these cells by fluorescence-activated cell sorting. Immunocytochemistry and qPCR analyses on freshly isolated ALDH(+) cells reveal an enrichment in cells expressing liver stem cell markers such as EpCAM, CK19, CD133, and Sox9. In culture, the ALDH(+) population can give rise to functional hepatocyte-like cells as illustrated by albumin and urea secretion and cytochrome P450 activity. ALDH1A1 expression can be detected in canals of Hering and bile duct epithelial cells and is increased on liver injury. Finally, we showed that the isolation and differentiation toward hepatocyte-like cells of LPCs with high ALDH activity is also successfully applicable to human liver samples. CONCLUSION: High ALDH activity is a feature of LPCs that can be taken advantage of to isolate these cells from untreated mouse as well as human liver tissues. This novel protocol is practically relevant, because it provides an easy and nontoxic method to isolate liver stem cells from normal tissue for potential therapeutic purposes.