Therapy For Liver Disease Research Articles

Therapeutic Pipeline in Alcohol-Associated Liver Disease.

Alcohol-associated liver disease is a leading cause of mortality and morbidity worldwide. Patients with alcohol-associated liver disease are often diagnosed at advanced stage and disease spectrum including alcoholic hepatitis, a severe manifestation with a high short-term mortality. Corticosteroid, recommended first-line treatment for patients with alcoholic hepatitis, is a very suboptimal treatment. Although the use of early liver transplantation has increased with consistent benefit in select patients with alcoholic hepatitis, its use remains heterogeneous worldwide due to lack of uniform selection criteria. Over the last decade, several therapeutic targets have evolved of promise with ongoing clinical trials in patients with cirrhosis and alcoholic hepatitis. Even with availability of effective medical therapies for alcohol-associated liver disease, long-term outcome depends on abstinence from alcohol use in any spectrum of alcohol-associated liver disease. However, alcohol use disorder treatment remains underutilized due to several barriers even in patients with advanced disease. There is an urgent unmet need to implement and promote integrated multidisciplinary care model with hepatologists and addiction experts to provide comprehensive management for these patients. In this review, we will discuss newer therapies targeting liver disease and therapies targeting alcohol use disorder in patients with alcohol-associated liver disease.

Selective Inhibition of Organic Cation Transporter 1 by Benzoylpaeoniflorin Attenuates Hepatic Lipid Accumulation through AMPK Activation.

Organic cation transporter 1 (OCT1) is a liver-specific transporter and plays an essential role in drug disposition and hepatic lipid metabolism. Therefore, inhibition of OCT1 may not only lead to drug-drug interactions but also represent a potential therapy for fatty liver diseases. In this study, we systematically investigated the inhibitory effect of 200 natural products on OCT1-mediated uptake of 4,4-dimethylaminostyryl-N-methylpyridinium (ASP+) and identified 10 potent OCT1 inhibitors. The selectivity of these inhibitors over OCT2 was evaluated using both in vitro uptake assays and in silico molecular docking analyses. Importantly, benzoylpaeoniflorin was identified as the most potent OCT1 inhibitor with the highest selectivity over OCT2. Additionally, benzoylpaeoniflorin prevented lipid accumulation in hepatocytes, with concomitant activation of AMPK and down-regulation of lipogenic genes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). To conclude, our findings are of significant value in understanding OCT1-based natural product-drug interactions and provide a natural source of OCT1 inhibitors which may hold promise for treating fatty liver diseases.

Current Therapeutic Options and Potential of Mesenchymal Stem Cell Therapy for Alcoholic Liver Disease

Alcoholic liver disease (ALD) is a globally prevalent chronic liver disease caused by chronic or binge consumption of alcohol. The therapeutic efficiency of current therapies for ALD is limited, and there is no FDA-approved therapy for ALD at present. Various strategies targeting pathogenic events in the progression of ALD are being investigated in preclinical and clinical trials. Recently, mesenchymal stem cells (MSCs) have emerged as a promising candidate for ALD treatment and have been tested in several clinical trials. MSC-released factors have captured attention, as they have the same therapeutic function as MSCs. Herein, we focus on current therapeutic options, recently proposed strategies, and their limitations in ALD treatment. Also, we review the therapeutic effects of MSCs and those of MSC-related secretory factors on ALD. Although accumulating evidence suggests the therapeutic potential of MSCs and related factors in ALD, the mechanisms underlying their actions in ALD have not been well studied. Further investigations of the detailed mechanisms underlying the therapeutic role of MSCs in ALD are required to expand MSC therapies to clinical applications. This review provides information on current or possible treatments for ALD and contributes to our understanding of the development of effective and safe treatments for ALD.

Extracellular Vesicles as Biomarkers in Liver Disease.

Extracellular vesicles (EVs) are membrane-derived vesicles released by a variety of cell types, including hepatocytes, hepatic stellate cells, and immune cells in normal and pathological conditions. Depending on their biogenesis, there is a complex repertoire of EVs that differ in size and origin. EVs can carry lipids, proteins, coding and non-coding RNAs, and mitochondrial DNA causing alterations to the recipient cells, functioning as intercellular mediators of cell-cell communication (auto-, para-, juxta-, or even endocrine). Nevertheless, many questions remain unanswered in relation to the function of EVs under physiological and pathological conditions. The development and optimization of methods for EV isolation are crucial for characterizing their biological functions, as well as their potential as a treatment option in the clinic. In this manuscript, we will comprehensively review the results from different studies that investigated the role of hepatic EVs during liver diseases, including non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic liver disease, fibrosis, and hepatocellular carcinoma. In general, the identification of patients with early-stage liver disease leads to better therapeutic interventions and optimal management. Although more light needs to be shed on the mechanisms of EVs, their use for early diagnosis, follow-up, and prognosis has come into the focus of research as a high-potential source of 'liquid biopsies', since they can be found in almost all biological fluids. The use of EVs as new targets or nanovectors in drug delivery systems for liver disease therapy is also summarized.

Farnesoid X receptor antagonizes macrophage-dependent licensing of effector T lymphocytes and progression of sclerosing cholangitis.

Immune-mediated bile duct epithelial injury and toxicity of retained hydrophobic bile acids drive disease progression in fibrosing cholangiopathies such as biliary atresia or primary sclerosing cholangitis. Emerging therapies include pharmacological agonists to farnesoid X receptor (FXR), the master regulator of hepatic synthesis, excretion, and intestinal reuptake of bile acids. Unraveling the mechanisms of action of pharmacological FXR agonists in the treatment of sclerosing cholangitis (SC), we found that intestinally restricted FXR activation effectively reduced bile acid pool size but did not improve the SC phenotype in MDR2-/- mice. In contrast, systemic FXR activation not only lowered bile acid synthesis but also suppressed proinflammatory cytokine production by liver-infiltrating inflammatory cells and blocked progression of hepatobiliary injury. The hepatoprotective activity was linked to suppressed production of IL1β and TNFα by hepatic macrophages and inhibition of TH1/TH17 lymphocyte polarization. Deletion of FXR in myeloid cells caused aberrant TH1 and TH17 lymphocyte responses in diethoxycarbonyl-1,4-dihydrocollidine-induced SC and rendered these mice resistant to the anti-inflammatory and liver protective effects of systemic FXR agonist treatment. Pharmacological FXR activation reduced IL1β and IFNγ production by liver- and blood-derived mononuclear cells from patients with fibrosing cholangiopathies. In conclusion, we demonstrate FXR to control the macrophage-TH1/17 axis, which is critically important for the progression of SC. Hepatic macrophages are cellular targets of systemic FXR agonist therapy for cholestatic liver disease.

In vivo phosphorus magnetic resonance spectroscopic imaging of the whole human liver at 7 T using a phosphorus whole-body transmit coil and 16-channel receive array: Repeatability and effects of principal component analysis-based denoising.

Quantitative three-dimensional (3D) imaging of phosphorus (31 P) metabolites is potentially a promising technique with which to assess the progression of liver disease and monitor therapy response. However, 31 P magnetic resonance spectroscopy has a low sensitivity and commonly used 31 P surface coils do not provide full coverage of the liver. This study aimed to overcome these limitations by using a 31 P whole-body transmit coil in combination with a 16-channel 31 P receive array at 7 T. Using this setup, we determined the repeatability of whole-liver 31 P magnetic resonance spectroscopic imaging (31 P MRSI) in healthy subjects and assessed the effects of principal component analysis (PCA)-based denoising on the repeatability parameters. In addition, spatial variations of 31 P metabolites within the liver were analyzed. 3D 31 P MRSI data of the liver were acquired with a nominal voxel size of 20 mm isotropic in 10 healthy volunteers twice on the same day. Data were reconstructed without denoising, and with PCA-based denoising before or after channel combination. From the test-retest data, repeatability parameters for metabolite level quantification were determined for 12 31 P metabolite signals. On average, 31 P MR spectra from 100 ± 25 voxels in the liver were analyzed. Only voxels with contamination from skeletal muscle or the gall bladder were excluded and no voxels were discarded based on (low) signal-to-noise ratio (SNR). Repeatability for most quantified 31 P metabolite levels in the liver was good to excellent, with an intrasubject variability below 10%. PCA-based denoising increased the SNR ~ 3-fold, but did not improve the repeatability for mean liver 31 P metabolite quantification with the fitting constraints used. Significant spatial heterogeneity of various 31 P metabolite levels within the liver was observed, with marked differences for the phosphomonoester and phosphodiester metabolites between the left and right lobe. In conclusion, using a 31 P whole-body transmit coil in combination with a 16-channel 31 P receive array at 7 T allowed 31 P MRSI acquisitions with full liver coverage and good to excellent repeatability.

Biomimetic Total Synthesis and the Biological Evaluation of Natural Product (-)-Fargesone A as a Novel FXR Agonist.

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, plays an important role in maintaining or reversing metabolic homeostasis during the development of liver diseases. However, developing FXR modulators to intervene in FXR-related diseases is still an unmet clinical need. Therefore, it is significant to develop novel small-molecule agonists for drug discovery targeting FXR. Through a high-throughput chemical screen and follow-up biological validations, we first identified the natural product Fargesone A (FA) as a potent and selective FXR agonist. The limited, variable supply of FA from natural product isolation, however, has impeded its biological exploration and potential drug development. Accordingly, we have developed a biomimetic and scalable total synthesis of FA in nine steps that provides a solution to the supply of FA. Enabled by chemical synthesis, the in vivo efficacy of FA has been further investigated. The results showed that FA alleviates hepatocyte lipid accumulation and cell death in an FXR-dependent manner. Moreover, treatment of bile duct ligation (BDL)-induced liver disorder with FA ameliorates pathological features in mice. Therefore, our work lays the foundation to develop new small-molecule FXR agonists as a potential therapy for liver diseases.

Generation of Hepatocytes and Nonparenchymal Cell Codifferentiation System from Human-Induced Pluripotent Stem Cells.

To date, hepatocytes derived from human-induced pluripotent stem cells (hiPSC) provide a potentially unlimited resource for clinical application and drug development. However, most hiPSC-derived hepatocyte-like cells initiated differentiation from highly purified definitive endoderm, which are insufficient to accurately replicate the complex regulation of signals among multiple cells and tissues during liver organogenesis, thereby displaying an immature phenotypic and short survival time in vitro. Here, we described a protocol to achieve codifferentiation of endoderm-derived hepatocytes and mesoderm-derived nonparenchymal cells by the inclusion of BMP4 into hepatic differentiation medium, which has a beneficial effect on the hepatocyte maturation and lifespan in vitro. Our codifferentiation system suggests the important role of nonparenchymal cells in liver organogenesis. Hopefully, these hepatocytes described here provide a promising approach in the therapy of liver diseases.

Selective Use of Pulmonary Vasodilators in Patients with Fontan Physiology.

Fontan-associated liver disease is a well-known sequela following the Fontan procedure for patients living with single-ventricle heart disease. Pulmonary vasodilators, such as phosphodiesterase type 5 inhibitors, have emerged as a potential therapeutic option for lowering central venous pressures by reducing pulmonary vascular resistance. We performed a single-center retrospective review of Fontan patients who were placed on pulmonary vasodilator therapy with prehemodynamic and posthemodynamic, MR elastography, and histologic assessments. A total of 125 patients with Fontan circulation underwent surveillance with cardiac catheterization during the review period. Fifty-three (42%) patients who did not have increased end-diastolic pressures at the time of cardiac catheterization were started on phosphodiesterase type 5 inhibitor therapy. Nine patients (17%) underwent posttherapy follow-up catheterization. The mean Fontan pressure decreased from 15.4 ± 3.3 mmHg to 13.3 ± 2.5 mmHg (p=0.026), after initiation of pulmonary vasodilatory therapy. There was no change in end-diastolic pressure, transpulmonary gradient, wedge pressure, pulmonary vascular resistance, cardiac index, or saturation. Eleven patients (21%) underwent pretherapy MR elastography testing with posttherapy follow-up MR elastography. We found no improvement in liver stiffness score following the application of pulmonary vasodilators. Three patients underwent pretherapy and posttherapy liver biopsies, with variable histological changes observed within the hepatic parenchyma. These data demonstrate indeterminate results for the selective use of pulmonary vasodilators but highlight the need for large prospective randomized control trials of pulmonary vasodilator therapies to fully assess the benefit of such therapies in Fontan-associated liver disease.

IL-2 availability regulates the tissue specific phenotype of murine intra-hepatic Tregs.

CD4+CD25+Foxp3+ Tregs are known to acquire tissue-specific features and exert cytoprotective and regenerative functions. The extent to which this applies to liver-resident Tregs is unknown. In this study, we aimed to explore the phenotypic and functional characteristics of adult murine liver resident Tregs during homeostasis. Additionally, we investigated their role in ameliorating liver inflammation and tissue damage. Quantification of Foxp3+CD4+CD25+ cells comparing different tissues showed that the liver contained significantly fewer resident Tregs. A combination of flow cytometry phenotyping and microarray analysis of intra-hepatic and splenic Tregs under homeostatic conditions revealed that, although intra-hepatic Tregs exhibited the core transcriptional Treg signature, they expressed a distinct transcriptional profile. This was characterized by reduced CD25 expression and increased levels of pro-inflammatory Th1 transcripts Il1b and Ifng. In vivo ablation of Tregs in the Foxp3-DTR mouse model showed that Tregs had a role in reducing the magnitude of systemic and intra-hepatic inflammatory responses following acute carbon tetrachloride (CCl₄) injury, but their absence did not impact the development of hepatocyte necrosis. Conversely, the specific expansion of Tregs by administration of IL-2 complexes increased the number of intra-hepatic Tregs and significantly ameliorated tissue damage following CCl₄ administration in C57BL/6 mice. The cytoprotective effect observed in response to IL-2c was associated with the increased expression of markers known to regulate Treg suppressive function. Our results offer insight into the transcriptome and complex immune network of intra-hepatic Tregs and suggest that strategies capable of selectively increasing the pool of intra-hepatic Tregs could constitute effective therapies in inflammatory liver diseases.

Penthorum chinense Pursh polysaccharide induces a mitochondrial-dependent apoptosis of H22 cells and activation of immunoregulation in H22 tumor-bearing mice

Penthorum chinense Pursh has been widely used as a Chinese traditional functional food for liver disease prevention and therapy. A novel cold-water soluble polysaccharide (PCP-4) has been prepared from Penthorum chinense Pursh at 4 °C. PCP-4 contained both α and β configurations and mainly consisted of galactose, arabinose, galacturonic acid, rhamnose, and glucose (molar ratio: 6.38:5.26:1.69:1:0.87) with an average molecular weight of 1.83 × 106 Da. In vitro experiments results demonstrated that PCP-4 effectively inhibits the proliferation of H22 cells and blocks cells at S phase in a dose-dependent manner. Otherwise, PCP-4-treated cells exhibited typical morphological features of apoptotic cells. Combined the results from western blot analysis, Annexin V-FITC/PI staining, Rhodamine 123 staining and DCFH-DA staining, PCP-4 was found to induce H22 cell apoptosis in vitro via the mitochondrial signaling pathway. The results of in vivo experiments suggested that PCP-4 significantly suppresses xenograft tumor growth by protecting immune organs, enhancing immune cells functionality, and inducing apoptosis. Overall, these results clearly show that Penthorum chinense Pursh polysaccharide serves as a valuable natural product for hepatocellular carcinoma therapy.

Morphometric parameters of sinusoidal capillaries, interlobular veins and interlobular arteries at different stages of experimental liver fibrosis

The aim of the study was to conduct a morphometric analysis of sinusoidal capillaries, interlobular veins and interlobular arteries of the liver at different stages of experimental fibrosis in order to investigate relationship between fibrogenesis and angiogenesis and identify new therapeutic targets. Material and methods. The study included male Wistar rats. Liver fibrosis with transformation into cirrhosis was induced with a solution of thioacetamide, which was injected into the stomach, dosage 200 mg/kg of animal body weight, using a probe 2 times a week. The rats of the experimental groups were withdrawn from the experiment in 3, 5, 7, 9, 11, 13, 15, and 17 weeks, and the intact ones - at the end of the experiment. To identify connective tissue, liver sections were stained according to the Mallory method. Immunohistochemical examination was performed on paraffin sections using a monoclonal mouse antibody to CD31. The number of sinusoidal capillaries, interlobular arteries, and interlobular veins was counted in three non-overlapping fields of view of each histological section using an OLYMPUS BX51 microscope at a 40× objective magnification. Results. The results obtained supported a statistically significant decreased number of sinusoidal capillaries (p=0,0000) and an increased number of interlobular veins (p=0,0000) in animals of the experimental group compared with the control group. There were no differences in the number of interlobular arteries (p=0,9999). Pronounced angiogenesis in the portal zones and connective tissue septa was associated with an increased amount of the connective tissue during liver fibrogenesis. Conclusion. In the future, angiogenesis modulation should be differentiated and directed towards antifibrotic, anti-inflammatory or autoimmune therapy in chronic liver diseases.

Metabolic basis for substantiation of nutrition therapy in chronic liver diseases

Over the past 10 years, an in-depth study of metabolic processes in chronic liver diseases and the rapid development of new technologies in the production of macro- and nutrients, pharmacological nutrients in clinical dietetics and nutritional science have led to the creation of fundamentally new approaches in nutritional therapy for these diseases. It is recognized that dietary interventions for chronic liver diseases should not only follow the general recommendations indicated as the need for energy and protein, but also contribute to the normalization of the amino acid composition of the blood, the metabolism of macro- and microelements, vitamins, etc. Methods of dietary correction are of particular importance hepatic encephalopathy, as well as the use of micro- and pharmacological nutrients for therapeutic purposes.

Prospects for the use of hepatotropic LOLA therapy in veterinary practice

Relevance. There are enough works devoted to the LOLA therapy in treatment ofhepatopathy of various origins, however, these works are devoted to research in medicine or in laboratory studies of animals.Methods. For inclusion in the experiment, screening studies of the blood plasma of horses for the level of gamma-glutamyltransferase were carried out. The inclusion criteria was the value of the activity of glutamyltransferase in blood serum more than 22 U/l (upper limit of the reference interval). As a result of screening studies, 20 animals for inclusion in the experiment were identified, which were divided into two parity groups. The second group received L-ornithine L-aspartate as hepatotropic therapy at a dosage of 0.05 g/kg of body weight orally (in the form of a powder, previously dissolved in water) once a day for 28 days.Results. On the 7th day of the experiment, the GGT level decreased by an average of 5.63% compared to the control, on the 14th day — by 12.45%, on the 21st day — by 15.95%, at the end of the experiment — by 20.09%. Beginning on day 14 of therapy the amount of GGT in the experimental group reached reference values.Clinical trial data support l the thesis that LOLA has hepatoprotective properties in cases of hepatopathy of various origins, which is proved by a decrease in the level of GGT in the blood serum. Larger studies with a variety of designs, as well as comparison with other organ-specific measures, are needed to confirm these initial results. Thus, the use of LOLA hepatotropic therapy in liver diseases receives a new justification for use in veterinary medicine.

Bifendate inhibits autophagy at multiple steps and attenuates oleic acid-induced lipid accumulation

Some traditional Chinese medicines exert roles in the therapy of liver diseases by modulating autophagy. Bifendate (DDB), a synthetic intermediate of Schisandrin C extracted from Schisandrae chinensis, is clinically used to treat hepatitis in China. While DDB is a positive control to research some potential hepatoprotective agents, its related molecular mechanisms are unknown. In this study, we show that DDB inhibited autophagosome−lysosome fusion, lysosome acidification and autophagic lysosome reformation. Moreover, DDB attenuated oleic acid-induced lipid droplet accumulation. These findings reveal the effects of DDB on the autophagy-related processes and lysosomal function, and also provide a possibility to understand the bioactivity mechanism of DDB in the future.

Registered Trials of Artificial Intelligence Conducted on Chronic Liver Disease: A Cross-Sectional Study on ClinicalTrials.gov.

Background Artificial intelligence (AI) has been widely applied in the diagnosis and therapy of chronic liver disease (CLD), but there is currently little insight into the trials registered on ClinicalTrials.gov. Thus, this cross-sectional study was focused on analyzing the progress in the use of AI in CLD. Methods Registered trials of AI applied in CLD on ClinicalTrials.gov were searched firstly. All available information was downloaded to Excel (Microsoft Excel, Rong, Rong, China), and duplicates were removed. We extracted the data of the included trials, then analyzed the characteristics of them finally. Results Up to the 27th of May 2021, 6835 trials were identified following an initial search, and 20 registered trials were included after screening for inclusion and exclusion criteria. Among those trials, hepatocellular carcinoma (HCC, 40.0%) and nonalcoholic fatty liver disease (NAFLD, 20.0%) were the most widely applied CLDs for AI. Trials started in 2013 until 2021, with 17 trials (85%) registered after 2016. There was a large trend in trial enrolment, with 40% of them including samples more than 500. Five trials (25%) have been completed, but only one of these had available results. The most frequent sponsors and collaborators were both hospitals at 55%, followed by universities at 35% and institutes at 11%, respectively. Of the 20 trials included, 35% (7 trials) were interventional trials and 65% (13 trials) were observational trials. Among 7 interventional trials, most trials were for diagnosis purpose (42.86%, 3 trials); 4 trials (57.14%) were randomized; 3 trials (42.86%) applied behavioral intervention, 1 trial (14.29%) was in device intervention, 2 trials (28.57%) were in diagnostic test, and 1 trial intervention was unknown. Among 13 observational trials, 8 (61.54%) were cohort studies; 6 (46.15%) were prospective studies, 4 (30.77%) were retrospective studies, 2 (15.38%) were cross-sectional studies, and 1 (7.69%) did not involve a temporal perspective. Conclusion The study is the first to focus on AI registration trials in CLD, which will aid relevant scholars in understanding the current state of the subject. This study demonstrates that additional research on AI used in the diagnosis and treatment of CLD is required, and timely publication of accessible results from registered trials is essential.

Exosome-mediated delivery of Cas9 ribonucleoprotein complexes for tissue-specific gene therapy of liver diseases.

CRISPR-Cas9 gene editing has emerged as a powerful therapeutic technology, but the lack of safe and efficient in vivo delivery systems, especially for tissue-specific vectors, limits its broad clinical applications. Delivery of Cas9 ribonucleoprotein (RNP) owns competitive advantages over other options; however, the large size of RNPs exceeds the loading capacity of currently available delivery vectors. Here, we report a previously unidentified genome editing delivery system, named exosomeRNP, in which Cas9 RNPs were loaded into purified exosomes isolated from hepatic stellate cells through electroporation. ExosomeRNP facilitated effective cytosolic delivery of RNP in vitro while specifically accumulated in the liver tissue in vivo. ExosomeRNP showed vigorous therapeutic potential in acute liver injury, chronic liver fibrosis, and hepatocellular carcinoma mouse models via targeting p53 up-regulated modulator of apoptosis (PUMA), cyclin E1 (CcnE1), and K (lysine) acetyltransferase 5 (KAT5), respectively. The developed exosomeRNP provides a feasible platform for precise and tissue-specific gene therapies of liver diseases.

The gut microbiota-bile acid axis: A potential therapeutic target for liver fibrosis.

Liver fibrosis involves the proliferation and deposition of extracellular matrix on liver tissues owing to various etiologies (including viral, alcohol, immune, and metabolic factors), ultimately leading to structural and functional abnormalities in the liver. If not effectively treated, liver fibrosis, a pivotal stage in the path to chronic liver disease, can progress to cirrhosis and eventually liver cancer; unfortunately, no specific clinical treatment for liver fibrosis has been established to date. In liver fibrosis cases, both the gut microbiota and bile acid metabolism are disrupted. As metabolites of the gut microbiota, bile acids have been linked to the progression of liver fibrosis via various pathways, thus implying that the gut microbiota–bile acid axis might play a critical role in the progression of liver fibrosis and could be a target for its reversal. Therefore, in this review, we examined the involvement of the gut microbiota–bile acid axis in liver fibrosis progression to the end of discovering new targets for the prevention, diagnosis, and therapy of chronic liver diseases, including liver fibrosis.

Human duodenal submucosal glands contain a defined stem/progenitor subpopulation with liver-specific regenerative potential

Common precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum. Human duodena were obtained from organ donors, and duodenal submucosal gland cells were isolated after removal of the mucosa layer. Cells were cultured on plastic or as organoids and were transplanted into severe combined immunodeficient (SCID) mouse livers. In situ studies of submucosal glands in the human duodenum revealed cells expressing stem/progenitor cell markers that had unique phenotypic traits distinguishable from intestinal crypt cells. Genetic signature studies indicated that the cells are closer to biliary tree stem cells and to definitive endodermal cells than to adult hepatocytes, supporting the interpretation that they are endodermal stem/progenitor cells. Invitro, human duodenal submucosal gland cells demonstrated clonal growth, capability to form organoids, and ability to acquire functional hepatocyte traits. Invivo, transplanted cells engrafted into the livers of immunocompromised mice and differentiated to mature liver cells. In an experimental model of fatty liver, human duodenal submucosal gland cells were able to rescue hosts from liver damage by supporting repopulation and regeneration of the liver. A cell population with clonal growth and organoid formation capability, which has liver differentiation potency invitro and invivo in murine experimental models, is present within adult duodenal submucosal glands. These cells can be isolated, do not require reprogramming, and thus could potentially represent a novel cell source for regenerative medicine of the liver. Cell therapies for liver disease could represent an option to support liver function, but the identification of sustainable and viable cell sources is critical. Here, we describe a cell population with organoid formation capability and liver-specific regenerative potential in submucosal glands of the human duodenum. Duodenal submucosal gland cells are isolated from adult organs, do not require reprogramming, and could rescue hepatocellular damage in preclinical models of chronic, but not acute, liver injury. Duodenal submucosal gland cells could represent a potential candidate cell source for regenerative medicine of the liver, but the determination of cell dose and toxicity is needed before clinical testing in humans.

Integrative network analysis of circular RNAs reveals regulatory mechanisms for hepatic specification of human iPSC-derived endoderm

BackgroundHuman-induced pluripotent stem cell (hiPSC)-derived functional hepatic endoderm (HE) is supposed to be an alternative option for replacement therapy for end-stage liver disease. However, the high heterogeneity of HE cell populations is still challenging. Hepatic specification of definitive endoderm (DE) is an essential stage for HE induction in vitro. Recent studies have suggested that circular RNAs (circRNAs) determine the fate of stem cells by acting as competing endogenous RNAs (ceRNAs). To date, the relationships between endogenous circRNAs and hepatic specification remain elusive.MethodsThe identities of DE and HE derived from hiPSCs were determined by qPCR, cell immunofluorescence, and ELISA. Differentially expressed circRNAs (DEcircRNAs) were analysed using the Arraystar Human circRNA Array. qPCR was performed to validate the candidate DEcircRNAs. Intersecting differentially expressed genes (DEGs) of the GSE128060 and GSE66282 data sets and the DEcircRNA-predicted mRNAs were imported into Cytoscape for ceRNA networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were involved in the enrichment analysis. Hepatic markers and Wnt/β-catenin were detected in hsa_circ_004658-overexpressing cells by western blotting. Dual-luciferase reporter assays were used to evaluate the direct binding among hsa_circ_004658, miRNA-1200 and CDX2. DE cells were transfected with miR-1200 mimics, adenovirus containing CDX2, and Wnt/β-catenin was detected by western blotting.ResultshiPSC-derived DE and HE were obtained at 4 and 9 days after differentiation, as determined by hepatic markers. During hepatic specification, 626 upregulated and 208 downregulated DEcircRNAs were identified. Nine candidate DEcircRNAs were validated by qPCR. In the ceRNA networks, 111 circRNA–miRNA–mRNA pairs were involved, including 90 pairs associated with hsa_circ_004658. In addition, 53 DEGs were identified among the intersecting mRNAs of the GSE128060 and GSE66282 data sets and the hsa_circ_004658-targeted mRNAs. KEGG and GO analyses showed that the DEGs associated with hsa_circ_004658 were mainly enriched in the WNT signalling pathway. Furthermore, hsa_circ_004658 was preliminarily verified to promote hepatic specification, as determined by hepatic markers (AFP, ALB, HNF4A, and CK19) (p < 0.05). This promotive effect may be related to the inhibition of the Wnt/β-catenin signalling pathway (detected by β-catenin, p-β-catenin, and TCF4) when hsa_circ_004658 was overexpressed (p < 0.05). Dual-luciferase reporter assays showed that there were binding sites for miR-1200 in the hsa_circ_004658 sequence, and confirmed the candidate DEG (CDX2) as a miR-1200 target. The level of miR-1200 decreased and the level of CDX2 protein expression increased when hsa_circ_004658 was overexpressed (p < 0.05). In addition, the results showed that CDX2 may suppress the Wnt/β-catenin signalling during hepatic specification (p < 0.05).ConclusionsThis study analysed the profiles of circRNAs during hepatic specification. We identified the hsa_circ_004658/miR-1200/CDX2 axis and preliminarily verified its effect on the Wnt/β-catenin signalling pathway during hepatic specification. These results provide novel insight into the molecular mechanisms involved in hepatic specification and could improve liver development in the future.Graphical