Hepatocyte Differentiation Research Articles

Adenine base editing and prime editing of chemically derived hepatic progenitors rescue genetic liver disease.

DNA base editors and prime editing technology enable therapeutic in situ correction of disease-causing alleles. These techniques could have broad applications for exvivo editing of cells prior to transplantation in a range of diseases, but it is critical that the target population is efficiently modified and engrafts into the host. Chemically derived hepatic progenitors (CdHs) are a multipotent population capable of robust engraftment and hepatocyte differentiation. Here we reprogrammed hepatocytes from a mouse model of hereditary tyrosinemia type 1 (HT1) into expandable CdHs and successfully corrected the disease-causing mutation using both adenine base editors (ABEs) and prime editors (PEs). ABE- and PE-corrected CdHs repopulated the liver with fumarylacetoacetate hydrolase-positive cells and dramatically increased survival of mutant HT1 mice. These results demonstrate the feasibility of precise gene editing in transplantable cell populations for potential treatment of genetic liver disease.

PFOA Exposure Prior to Hepatocyte Differentiation Leads to Gene Expression Changes Implicated in Non-Alcoholic Fatty Liver Disease

Background: Perfluorooctanoic acid (PFOA), is a persistent fluorinated compound with oil and water repelling properties found in cookware, food packaging and municipal water systems. Adult animals exposed to PFOA develop hepatomegaly, fatty liver, peroxisome proliferation, and immunotoxicity. Rodents exposed to PFCs in utero have altered hepatic lipid metabolism, increased hepatic de novo lipogenesis and susceptibility to non-alcoholic fatty liver disease (NAFLD), but underlying molecular mechanisms remain unknown. With increasing rates of obesity, diabetes, and NAFLD it is critical to examine the mechanisms by which in utero exposure to PFOA contributes to the development of metabolic syndrome in offspring. Objective: To determine mechanism by which PFOA alters gene expression in undifferentiated hepatic progenitor cells. Design/methods: HepaRG cells, a human derived hepatocyte progenitor cell line, was treated with 0.5uM PFOA or vehicle for 48 hours followed by differentiation into hepatocytes. Total RNA was extracted using the RNeasy (Qiagen) [total RNA A260/280>2 and RNA integrity number >7 (Agilent Bioanalyzer)] to generate libraries with the Illumina TruSeq stranded total RNA kit. RNA-Seq was performed using 85 bp single-end read sequencing to generate >20 million reads per sample. RNAseq data was aligned to hg38 using STAR v2.6.1a and then quantified with featureCounts v1.6.2. DESeq2 identified differentially expressed genes via FDR (false discovery rate) after Bonferroni correction. Differentially expressed gene lists were used for Ingenuity Pathway Analysis (IPA) to identify pathways of biological significance.Results: PFOA treatment resulted in increased expression of transcription factors EGR1 (early growth response protein 1), NR4A1 (nuclear receptor Nur77), EGR2 (early growth response protein 2), KLF10 (Krueppel-like factor 10) and FOSL1 (Fos-related antigen 1), key genes linked to impaired hepatic insulin signaling, hepatic lipid metabolism, steatosis and fibrosis (fold change > 1.5; q <0.05). IPA identified enrichment of canonical pathways with biological relevance including hepatic fibrosis signaling, stellate cell activation, VDR/RXR/TR activation, and Type 2 diabetes mellitus signaling (p<0.01). Conclusion: Hepatocyte progenitor cells exposed to low dose PFOA for 48 hours prior to differentiation results in changes in expression of key metabolic genes linked to the development of NAFLD and enrichment of biologically relevant pathways associated with hepatic fibrosis and hepatocellular carcinoma. These results suggest that PFOA exposure in utero may have lasting effects on hepatic glucose and lipid metabolism after differentiation. Further studies are needed to characterize the longstanding metabolic effects of in utero PFOA exposure in offspring and the mechanisms driving the persistence of these changes.

Mesenchymal Stem Cells (MSCs) in Targeted Drug Delivery: Literature Review and Exploratory Data on Migrating and Differentiation Capacities of Bone MSCs into Hepatic Progenitor Cells.

Mesenchymal stem cells (MSCs), particularly bone MSCs (BMSCs) offer great potentials for targeted therapeutic applications owing to their migratory and differentiation capacities. Significant advances have been achieved in the differentiation of hepatocyte or hepatocyte-like cells both in vitro and in vivo. However, there is limited knowledge on the differentiation of BMSCs into bipotential hepatic progenitor cells or cholangiocyte. This study reviews the potentials and advances in using MSCs as vehicles for targeted drug delivery and proposes a new method for the induction of differentiation in rat BMSCs into hepatic progenitor cells in vitro and assesses the differential and migratory capacities. The BMSCs of Sprague Dawley (SD) rats were harvested from the femur and the tibiae of the rats. After isolation and culturing, BMSCs from Passage 1 were used for the study. The in vitro differentiation of the hepatic progenitor cells was performed using a 2-step induction approach after 5-day serum deprivation from the BMSCs and culturing in Dulbecco's modified eagle medium. Spontaneous in vitro differentiation of BMSCs was examined in the absence of growth factors for 15 days as control treatment. Hepatocytes differentiation was achieved by exposing the culture to collagen type I-coated plates. Cholangiocytes differentiation was achieved with replating the BMC-HepPCs on a layer of Matrigel. Immunofluorescence was conducted on twelve-well plates to determine cell differentiations. Real-Time Quantitative Reverse Transcription PCR (qRTPCR) was used to determine the total RNA extracted using the Trizol LS reagent. In the hepatocyte differentiation group, after periodic acid-schiff (PAS) staining for glycogen, inverted microscope was used to determine differentiations and undifferentiated BMC-HepPCs served as controls. The amount of low-density lipoprotein (LDL) uptake by the BMSCs-derived hepatocytes was assessed using fluorescence microscopy. The secretion of rat albumin was quantified using a quantitative ELISA kit. Differentiation induction is indicative of the sequential supplementation of sodium butyrate and cytokines, which are involved in the embryonic development of the mammalian liver. Hepatic progenitor cells, derived from bone marrow, can be differentiated bidirectionally in vitro into both hepatocyte and cholangiocyte cell-lines. The differentiated cells, including hepatic progenitor cells, hepatocytes, and bile duct-like cells, were identified and analyzed at mRNA and protein levels. Our findings show that BMSCs can be utilized as novel bipotential hepatic progenitor cells and thereby for hepatobiliary disease treatment or hepatobiliary tissue-engineering.

The Inside-Out of End-Stage Liver Disease: Hepatocytes are the Keystone.

Chronic liver injury results in cirrhosis and end-stage liver disease (ESLD) which represents a leading cause of death worldwide, affecting people in their most productive years of life. Medical therapy can extend life, but the only definitive treatment is liver transplantation (LT). However, LT remains limited by access to quality donor organs and suboptimal long-term outcomes. The degeneration from healthy-functioning livers to cirrhosis and ESLD involves a dynamic process of hepatocyte damage, diminished hepatic function, and adaptation. However, the mechanisms responsible for deterioration of hepatocyte function and ultimately hepatic failure in man are poorly understood. We review the current understanding of cirrhosis and ESLD as a dynamic process and outline the current mechanisms associated with the development of hepatic failure from the clinical manifestations to energy adaptations, regeneration, and regulation of nuclear transcription factors. A new generation of therapeutics could target stabilization of hepatocyte differentiation and function to avoid the need for transplantation in patients with cirrhosis and ESLD.

Hippo signaling pathway in companion animal diseases, an under investigated signaling cascade

The Hippo pathway is a highly conserved kinase cascade in mammals with the proteins YAP and TAZ as its most important downstream effectors that shuttle between cytoplasma and nucleus. It has a crucial role in processes such as embryogenesis, organ size control, homeostasis and tissue regeneration, where mechanosensing and/or cell-cell interactions are involved. As the pathway is associated with many essential functions in the body, its dysregulation is related to many diseases. In contrast to human pathology, a PubMed-search on Hippo, YAP/TAZ and companion animals (horse, equine, dog, canine, cat, feline) retrieved few publications. Because of its high level of functional conservation, it is anticipated that also in veterinary sciences aberrant Hippo YAP/TAZ signaling would be implicated in animal pathologies. Publications on Hippo YAP/TAZ in companion animals are mainly in cats and dogs and related to oncology. Here, we emphasize the important role of YAP/TAZ in liver diseases. First the liver has a remarkable regeneration capacity and a strict size control and the liver has a moderate liver cell renewal (homeostasis). The last years numerous papers show the importance of YAP/TAZ in hepatocellular carcinoma (HCC), hepatocyte differentiation and bile duct epithelial (BEC) cell survival. YAP/TAZ signaling is involved in activation of hepatic stellate cells crucial in fibrogenesis. The availability of drugs (e.g. verteporfin) targeting the YAP/TAZ pathway are described as is their potential usage in veterinary medicine. The aim of this overview is to stimulate researchers’ and clinicians’ interest in the potential role of Hippo YAP/TAZ signaling in veterinary medicine.

DNA methylation as a key epigenetic player for hepatoblastoma characterization

BackgroundHepatoblastoma (HB) is a rare embryonal liver tumor of children. Although intrinsic biological differences between tumors can affect prognosis, few groups have studied these differences. Given the recent increased attention to epigenetic mechanisms in the genesis and progression of these tumors, we aimed to classify HB samples according to the stages of liver development and DNA methylation machinery. Basic proceduresWe evaluated the expression of 24 genes associated with DNA methylation and stages of hepatocyte differentiation and global DNA methylation. Using bioinformatics tools and expression data, we propose a stratification model for HB. Main findingsTumors clustered into three groups that presented specific gene expression profiles of the panel of DNA methylation enzymes and hepatocyte differentiation markers. In addition to reinforcing these embryonal tumors’ molecular heterogeneity, we propose that a panel of 13 genes can stratify HBs (TET1, TET2, TET3, DNMT1, DNMT3A, UHRF1, ALB, CYP3A4, TDO2, UGT1A1, AFP, HNF4A, and FOXA2). DNA methylation machinery participates in the characterization of HBs, directly reflected in diverse DNA methylation content. The data suggested that a subset of HBs were similar to differentiated livers, with upregulation of mature hepatocyte markers, decreased expression of DNA methylation enzymes, and higher global methylation levels; these findings might predict worse outcomes. ConclusionsHBs are heterogeneous tumors. Despite using a small cohort of 21 HB samples, our findings reinforce that DNA methylation is a robust biomarker for this tumor type.

STRATEGIES FOR HEPATOCYTE DIFFERENTIATION DERIVED FROM INDUCED PLURIPOTENT STEM CELLS USING SPHEROIDS

<h3>Background/Objective</h3> One of the main cells in the liver is the hepatocyte, which regulates the metabolism of various nutrients and xenobiotics, mediates endocrine function and excretion of toxic substances. Hepatocytes function in vitro have been studied for many years using primary hepatocytes and immortalized cells. Nevertheless, these cells show phenotypic instability or loss of adult liver functions and altered expression of important transcription factors. As an alternative to obtain a stable genetic background with preserved function and in large quantities, hepatocytes are being generated through differentiation of induced pluripotent stem cells (iPSC). However, one of the main problems to be overcome is the phenotype maturation. In this context, the objective of this work was to compare two different hepatocyte differentiation protocols using spheroids derived from iPSC. <h3>Methods</h3> Immunofluorescence for OCT3/4 and Nanog was performed to characterized pluripotent phenotype of iPSC and hepatocyte differentiation was induced for 28 days (D28). Two plating strategies were adopted to form the definitive endoderm: iPSC-monolayers (M) and iPSC-spheroids (S). To induce definitive endoderm, activin A and CHIR99021 were added to both plating conditions for 5 days. Prior hepatic maturation, iPSC-monolayers were also aggregated to form spheroids (MS). Subsequently, HGF and SB431542 were added to MS and S medium for 23 days. Spheroids were evaluated by real time PCR and immunofluorescence for hepatocyte-specific markers at D28. <h3>Results</h3> At protein level, the iPSC showed a pluripotent character through the nuclear presence of OCT3/4 and NANOG. At the end of the differentiation protocol, S differentiation strategy exhibited contractile cells. Albumin and cardiac troponin T were detected into the same spheroid by immunofluorescence. The MS strategy showed no beating cells. Also, CYP3A4 and albumin were detected by immunofluorescence and detectable mRNA levels were expressed for HNF4α, transferrin and alpha-1 antitrypsin. <h3>Conclusions</h3> Hepatocyte differentiation using spheroids requires a monolayer endoderm formation step in order to avoid the presence contractile cells at the end of protocol. Moreover, spheroid cells exhibited a great mature hepatocyte phenotype and can be used as a powerful tool for cell therapy, liver bioengineering, studies of drug efficacy and toxicity assays.

Update on Pathologic and Radiologic Diagnosis of Combined Hepatocellular-Cholangiocarcinoma.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a malignant primary liver carcinoma characterized by the unequivocal presence of both hepatocytic and cholangiocytic differentiation within the same tumor. Recent research has highlighted that cHCC-CCAs are more heterogeneous than previously expected. In the updated consensus terminology and WHO 2019 classification, "classical type" and "subtypes with stem-cell features" of the WHO 2010 classification are no longer recommended. Instead, it is recommended that the presence and percentages of various histopathologic components and stem-cell features be mentioned in the pathologic report. The new terminology and classification enable the exchange of clearer and more objective information about cHCC-CCAs, facilitating multi-center and multi-national research. However, there are limitations to the diagnosis of cHCC-CCA by imaging and biopsy. cHCC-CCAs showing typical imaging findings of HCC could be misdiagnosed as HCC and subjected to inappropriate treatment, if other clinical findings are not sufficiently considered. cHCC-CCAs showing at least one of the CCA-like imaging features or unusual clinical features should be subjected to biopsy. There may be a sampling error for the biopsy diagnosis of cHCC-CCA. An optimized diagnostic algorithm integrating clinical, radiological, and histopathologic information of biopsy is required to resolve these diagnostic pitfalls.

Online monitoring of hiPSC expansion and hepatic differentiation in 3D culture by dielectric spectroscopy.

Hepatocyte-like cells derived from human-induced pluripotent stem cells (hiPSC-HLC) are expected to have important applications in drug screening and regenerative medicine. However, hiPSC-HLC are difficult to produce on a large-scale to obtain relevant numbers for such applications. The aim of this study was to implement a novel integrated strategy for scalable production of hiPSC-HLC and demonstrate the applicability of dielectric spectroscopy to monitor hiPSC expansion/differentiation processes. We cultured hiPSC as three-dimensional (3D) aggregates in stirred-tank bioreactors (STB) operated in perfusion with an in situ capacitance probe. Dissolved oxygen concentration and dilution rate were controlled along the process and after 5 days of cell expansion, the hepatic differentiation was integrated in sequential steps for 28 days. The hiPSC were able to grow as 3D aggregates and the expression of hepatic markers and albumin production after differentiation confirmed that hepatocyte differentiation improved when compared to 2D culture. These hiPSC-HLC exhibited functional characteristics of hepatocytes including glycogen storage and drug metabolization capacity. Our results also show a good correlation between the cell permittivity measured online and the aggregate biovolume measured by standard offline methods, demonstrating for the first time the potential of dielectric spectroscopy to monitor hiPSC expansion and differentiation in STB.

Molecular and morphological changes induced by ivosidenib correlate with efficacy in mutant-IDH1 cholangiocarcinoma.

Background: IDH1mutations occur in approximately13% of intrahepatic cholangiocarcinomas (IHCCs). The oral, targeted, mutant IDH1 (mIDH1) inhibitorivosidenib (AG-120) suppresses production of the oncometabolite D-2-hydroxyglutarate, promoting disease stabilization and improved progression-free survival (PFS) in mIDH1 IHCC. Materials & methods: Harnessing matched baseline and on-treatment biopsies, we investigate the potential mechanisms underlying ivosidenib's efficacy. Results: mIDH1 inhibition leads to decreased cytoplasm and expression of hepatocyte lineage markers in patients with prolonged PFS. These findings are accompanied by downregulation of biliary fate, cell cycle progression and AKT pathway activity. Conclusion: Ivosidenib stimulates a hepatocyte differentiation program in mIDH1 IHCC, a phenotype associated with clinical benefit. mIDH1 inhibition could be a paradigm for differentiation-based therapy in solid tumors. Clinicaltrial registration: NCT02073994 (ClinicalTrials.gov).

Hepatocyte-Specific Hepatocyte Nuclear Factor 4 Alpha (HNF4) Deletion Decreases Resting Energy Expenditure by Disrupting Lipid and Carbohydrate Homeostasis.

Hepatocyte nuclear factor 4 alpha (HNF4) is required for hepatocyte differentiation and regulates expression of genes involved in lipid and carbohydrate metabolism including those that control VLDL secretion and gluconeogenesis. Whereas previous studies have focused on specific genes regulated by HNF4 in metabolism, its overall role in whole-body energy utilization has not been studied. In this study, we used indirect calorimetry to determine the effect of hepatocyte-specific HNF4 deletion (HNF4-KO) in mice on whole-body energy expenditure (EE) and substrate utilization in fed, fasted, and high-fat diet (HFD) conditions. HNF4-KO had reduced resting EE during fed conditions and higher rates of carbohydrate oxidation with fasting. HNF4-KO mice exhibited decreased body mass caused by fat mass depletion despite no change in energy intake and evidence of positive energy balance. HNF4-KO mice were able to upregulate lipid oxidation during HFD, suggesting that their metabolic flexibility was intact. However, only hepatocyte-specific HNF4-KO mice exhibited significant reduction in basal metabolic rate and spontaneous activity during HFD. Consistent with previous studies, hepatic gene expression in HNF4-KO supports decreased gluconeogenesis and decreased VLDL export and hepatic -oxidation in HNF4-KO livers across all feeding conditions. Together, our data suggest that deletion of hepatic HNF4 increases dependence on dietary carbohydrates and endogenous lipids for energy during fed and fasted conditions by inhibiting hepatic gluconeogenesis, hepatic lipid export, and intestinal lipid absorption resulting in decreased whole-body energy expenditure. These data clarify the role of hepatic HNF4 on systemic metabolism and energy homeostasis.

Dual β-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-β and Hepatocyte Nuclear Factor 4α Signaling

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

Functional inhibition of Oct leads to HNF4α upregulation.

Organic cation transporters (human, OCT; mouse, Oct) are responsible for the intracellular uptake and detoxification of a broad spectrum of endogenous and exogenous substrates. The OCT1 gene SLC22A1 (human; mouse, Scl22a1) is transactivated by hepatocyte nuclear factor 4α (human, HNF4α; mouse, Hnf4α). HNF4α is a master regulator of hepatocyte differentiation and is frequently associated with hepatocellular carcinoma (HCC). In addition, the downregulation of HNF4α is associated with enhanced fibrogenesis. Our recent study revealed that hepatocarcinogenesis and fibrosis were enhanced with the loss of Oct3 (gene, Slc22a3). Notably, differences in Hnf4α expression, and in cholestasis and fibrosis were also detected in Oct3-knockout (FVB.Slc22a3tm10pb, Oct3-/-) mice. To the best of our knowledge, no data exists on an interaction between Oct3 and Hnf4α. We hypothesised that loss of Oct3 may have an impact on Hnf4α expression. In the present study, gene expression analyses were performed in liver tissue from untreated Oct3-/- and wild type (FVB, WT) mice. C57BL/6, Oct3-/- and WT mice were treated with pro-fibrotic carbon tetrachloride (CCl4) or thioacetamide (TAA) for 6 weeks to chemically induce liver fibrosis. Cholestasis-associated fibrosis was mechanically generated in Oct3-/- and WT mice by bile duct ligation (BDL). Finally, stably OCT1- and OCT3-transfected tumour cell lines and primary murine hepatocytes were treated with the non-selective OCT inhibitor quinine and Hnf4α expression was quantified by qPCR and immunofluorescence. The results revealed that Hnf4α is one of the top upstream regulators in Oct3-/- mice. Hnf4α mRNA expression levels were downregulated in Oct3-/- mice compared with in WT mice during cholestatic liver damage as well as fibrogenesis. The downregulation of Hnf4α mRNA expression in fibrotic liver tissue was reversible within 4 weeks. In stably OCT1- and OCT3-transfected HepG2 and HuH7 cells, and primary murine hepatocytes, functional inhibition of OCT led to the upregulation of Hnf4α mRNA expression. Hnf4α was revealed to be located in the cytosol of WT hepatocytes, whereas Oct3-/- hepatocytes exhibited nuclear Hnf4α expression. In conclusion, Hnf4α was downregulated in response to cholestasis and fibrosis, and functional inhibition of Oct may lead to the upregulation of Hnf4α.

PGC7 promotes tumor oncogenic dedifferentiation through remodeling DNA methylation pattern for key developmental transcription factors

Poorly differentiated tumors usually exhibit phenotypes similar to that of their developmental precursor cells. Tumor cells that acquire the lineage progenitor cells feature usually exploit developmental signaling to potentiate cancer progression. However, the underlying molecular events remain elusive. In this study, based on analysis of an in vitro hepatocyte differentiation model, the maternal factor PGC7 (also known as DPPA3, STELLA) was found closely associated with liver development and tumor differentiation in hepatocellular carcinoma (HCC). Expression of PGC7 decreased during hepatocyte maturation and increased progressively from well-differentiated HCCs to poorly differentiated HCCs. Whole-genome methylation sequencing found that PGC7 could induce promoter demethylation of genes related to development. Pathway-based network analysis indicated that downstream targets of PGC7 might form networks associated with developmental transcription factor activation. Overexpression of PGC7 conferred progenitor-like features of HCC cells both in vitro and in vivo. Mechanism studies revealed that PGC7 could impede nuclear translocation of UHRF1, and thus facilitate promoter demethylation of GLI1 and MYCN, both of which are important regulators of HCC self-renewal and differentiation. Depletion or inhibition of GLI1 effectively downregulated MYCN, abolished the effect of PGC7, and sensitized HCC cells to sorafenib treatment. In addition, we found a significant correlation of PGC7 with GLI1/MYCN and lineage differentiation markers in clinical HCC patients. PGC7 expression might drive HCC toward a “dedifferentiated” progenitor lineage through facilitating promoter demethylation of key developmental transcription factors; further inhibition of PGC7/GLI1/MYCN might reverse poorly differentiated HCCs and provide novel therapeutic strategies.

Hsa-miR-637 inhibits human hepatocyte proliferation by targeting Med1-interacting proteins

BackgroundRecent studies have shown that mediator complex subunit 1 (Med1) can significantly affect hepatocyte proliferation and differentiation. Acting as a tumor suppressor, microRNA-637 (hsa-miR-637) can inhibit the growth of hepatocarcinoma cells and further induce cell apoptosis. However, the function of hsa-miR-637 and its target genes during liver regeneration remains to be elucidated. MethodsThis study used co-immunoprecipitation (Co-IP) assay, transfection, luciferase reporter assay, functional assay by cell counting kit-8 (CCK-8), Annexin V-FITC/propidium iodide apoptosis assay, and quantitative polymerase chain reaction analysis of chromatin immunoprecipitation (ChIP) for analysis. ResultsHsa-miR-637 has been suggested to suppress the expression of two Med1-interacting nuclear receptors, identified as the peroxisome proliferator-activated receptor alpha (PPARA) and thyroid hormone receptor alpha (THRA) at the transcriptional and translational levels in the human liver HL-7702 cell line. The interaction between Med1 and PPARA/THRA in HL-7702 cells was then confirmed. The transcriptional repression of hsa-miR-637 on PPARA and THRA was also demonstrated. Moreover, hsa-miR-637 has been determined to suppress the proliferation of HL-7702 cells. Furthermore, cell cycle arrest of HL-7702 cells was induced by transfection of hsa-miR-637 at the S phase, but its apoptosis failed. Finally, PPARA was indicated to directly bind to the promoter of some transcription factors, like β-catenin, mouse double minute 2 (MDM2), and p53. ConclusionsThis study has confirmed that hsa-miR-637 plays an antiproliferative role during liver regeneration, which may contribute in understanding the regenerative process of the liver.

In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide

An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.

Synergistic effect of three-dimensionalcoculture and photobiomodulation therapy on vascularized liver spheroid formation by stem cells.

Despite studies reporting functional differentiation of liver cells, a three-dimensional, vascularized liver organ has yet to be developed from mesenchymal stem cells. We investigated whether treatment with photobiomodulation (PBM) before three-dimensional liver spheroid transplantation improved the recovery of liver function via stimulation of angiogenesis and hepatocyte differentiation. Liver spheroids composed of hepatic, endothelial,and mesenchymal cells were subjected to PBM therapy. To evaluate the in vivo therapeutic effect of the liver spheroids treated with PBM, phosphate-buffered saline, liver spheroid, and PBM-treated liver spheroid were transplanted into a damaged host liver using conventional chimeric mouse models. To further characterize the maturation of transplanted PBM-liver spheroid compared with the newly generated non-PBM-liver spheroid or human liver tissues, the expression profiles of mature liver signature genes were analyzed. Liver spheroids expressed hepatocyte growth factors, including vascular endothelial growth factor and angiogenic factors. The cells in liver spheroid compensated for the low viability and improved the function of hepatocytes. Here, we demonstrate the formation of vascularized and functional human liver spheroid from human adipose-derived stem cellsby transplantation of liver tissue created in vitro. Albumin secretion by PBM-treated liver spheroid was higher on Day 28 compared with liver spheroid-seeded transplant group. PBM-liver spheroids serve as individual vascularization units, promoting the simultaneous development of new microvascular networks at different locations inside the implanted tissue constructs. The vasculature in the liver spheroid transplants became functional by connecting to the host vessels within 48 h. These PBM-liver spheroids may be useful in designing artificial three-dimensional hepatic tissue constructs and in cell therapy with limited numbers of human hepatocytes.

Transcriptional and epigenetic landscape of Ca2+-signaling genes in hepatocellular carcinoma.

Calcium (Ca2+) signaling has a major role in regulating a wide range of cellular mechanisms, including gene expression, proliferation, metabolism, cell death, muscle contraction, among others. Recent evidence suggests that ~ 1600 genes are related to the Ca2+ signaling. Some of these genes' expression is altered in several pathological conditions, including different cancer types, and epigenetic mechanisms are involved. However, their expression and regulation in hepatocellular carcinoma (HCC) and the liver are barely known. Here, we aimed to explore the expression of genes involved in the Ca2+-signaling in HCC, liver regeneration, and hepatocyte differentiation, and whether their expression is regulated by epigenetic mechanisms such as DNA methylation and histone posttranslational modifications (HPM). Results show that several Ca2+-signaling genes' expression is altered in HCC samples; among these, a subset of twenty-two correlate with patients' survival. DNA methylation correlates with eight of these genes' expression, and Guadecitabine, a hypomethylating agent, regulates the expression of seven down-regulated and three up-regulated genes in HepG2 cells. The down-regulated genes displayed a marked decrease of euchromatin histone marks, whereas up-regulated genes displayed gain in these marks. Additionally, the expression of these genes is modulated during liver regeneration and showed similar profiles between in vitro differentiated hepatocytes and liver-derived hepatocytes. In conclusion, some components of the Ca2+-signaling are altered in HCC and displayed a correlation with patients' survival. DNA methylation and HMP are an attractive target for future investigations to regulate their expression. Ca2+-signaling could be an important regulator of cell proliferation and differentiation in the liver.

Lnc-RHL, a novel long non-coding RNA required for the differentiation of hepatocytes from human bipotent progenitor cells.

ObjectivesThe final stage of liver development is the production of hepatocytes and cholangiocytes (biliary epithelial cells) from bipotent hepatic progenitor cells. We used HepaRG cells, which are bipotent and able to differentiate into both hepatocytes and cholangiocytes, as a model to study the action of a novel lncRNA (lnc‐RHL) and its role in the regulation of bipotency leading to hepatocytes and cholangiocytes.Materials and MethodsDifferentiation of HepaRG cells was assessed by marker expression and morphology which revealed their ability to differentiate into hepatocytes and cholangiocytes (modelling the behaviour of hepatoblasts in vivo). Using a qRT‐PCR and RACE, we cloned a novel lncRNA (lnc‐RHL; regulator of hepatic lineages) that is upregulated upon HepaRG differentiation. Using inducible knockdown of lnc‐RHL concurrently with differentiation, we show that lnc‐RHL is required for proper HepaRG cell differentiation resulting in diminution of the hepatocyte lineage.ResultsHere, we report the discovery of lnc‐RHL, a spliced and polyadenylated 670 base lncRNA expressed from the 11q23.3 apolipoprotein gene cluster. lnc‐RHL expression is confined to hepatic lineages and is upregulated when bipotent HepaRG cells are caused to differentiate. HepaRG cells made deficient for lnc‐RHL have reduced ability to differentiate into hepatocytes, but retain their ability to differentiate into cholangiocytes.ConclusionsDeficiency for lnc‐RHL in HepaRG cells converts them from bipotent progenitor cells to unipotent progenitor cells with impaired ability to yield hepatocytes. We conclude that lnc‐RHL is a key regulator of bipotency in HepaRG cells.

Hypoxia-Induced miR-210 Overexpression Promotes the Differentiation of Human-Induced Pluripotent Stem Cells to Hepatocyte-Like Cells on Random Nanofiber Poly-L-Lactic Acid/Poly (ε-Caprolactone) Scaffolds.

An alternative treatment to liver transplantation includes the use of differentiated stem cells. Hypoxia has been shown to endow human-induced pluripotent stem cells (hiPSCs) with enhanced hepatic differentiation. We have investigated a new strategy for hepatocyte differentiation from hiPSCs using a three-step differentiation protocol with lentiviral overexpression of hypoxia-microRNA-210 of cells grown on a hybrid scaffold. We analyzed the transduction of the miR-210 lentiviral and definitive endoderm and pluripotency gene markers, including SRY-box 17 (SOX17), forkhead box A2 (FOXA2), and octamer-binding transcription factor 4 (OCT-4) by Real-Time PCR and fluorescent microscope. The scanning electron microscopy (SEM) examined the 3D cell morphological changes. Immunocytochemistry staining was used together with assays for aspartate aminotransferase, alanine aminotransferase, and urea secretion to analyze hepatocyte biomarkers and functional markers consisting of α-fetoprotein (AFP), low-density lipoprotein (LDL) uptake, fat accumulation, and glycogen. The flow cytometry analyzed the generation of reactive oxygen species (ROS). Compared to cells transfected with the blank lentiviral vectors as a control, overexpressing miR-210 was at higher levels in hiPSCs. The expression of endodermal genes and glycogen synthesis significantly increased in the differentiated lentiviral miR-210 cells without any differences regarding lipid storage level. Additionally, cells containing miR-210 showed a greater expression of ALB, LDL, AST, ALT, urea, and insignificant lower AFP and ROS levels after 18 days. However, SEM showed no significant differences between cells under the differentiation process and controls. In conclusion, the differentiation of hiPSCs to hepatocyte-like cells under hypoxia miR-210 may be a suitable method for cell therapy and regenerative medicine.