Deficiency of ASGR1 Alleviates Diet-Induced Systemic Insulin Resistance via Improved Hepatic Insulin Sensitivity.

Background: Insulin resistance (IR) is the key pathological basis of many metabolic disorders. Lack of asialoglycoprotein receptor 1 (ASGR1) decreased the serum lipid levels and reduced the risk of coronary artery disease. However, whether ASGR1 also participates in the regulatory network of insulin sensitivity and glucose metabolism remains unknown. Methods: The constructed ASGR1 knockout mice and ASGR1-/- HepG2 cell lines were used to establish the animal model of metabolic syndrome and the IR cell model by high-fat diet (HFD) or drug induction, respectively. Then we evaluated the glucose metabolism and insulin signaling in vivo and in vitro. Results: ASGR1 deficiency ameliorated systemic IR in mice fed with HFD, evidenced by improved insulin intolerance, serum insulin, and homeostasis model assessment of IR index, mainly contributed from increased insulin signaling in the liver, but not in muscle or adipose tissues. Meanwhile, the insulin signal transduction was significantly enhanced in ASGR1-/- HepG2 cells. By transcriptome analyses and comparison, those differentially expressed genes between ASGR1 null and wild type were enriched in the insulin signal pathway, particularly in phosphoinositide 3-kinase-AKT signaling. Notably, ASGR1 deficiency significantly reduced hepatic gluconeogenesis and glycogenolysis. Conclusion: The ASGR1 deficiency was consequentially linked with improved hepatic insulin sensitivity under metabolic stress, hepatic IR was the core factor of systemic IR, and overcoming hepatic IR significantly relieved the systemic IR. It suggests that ASGR1 is a potential intervention target for improving systemic IR in metabolic disorders.

Hepatic DKK1-driven steatosis is CD36 dependent.

Nonalcoholic fatty liver disease (NAFLD) is prevalent worldwide; about 25% of NAFLD silently progress into steatohepatitis, in which some of them may develop into fibrosis, cirrhosis and liver failure. However, few drugs are available for NAFLD, partly because of an incomplete understanding of its pathogenic mechanisms. Here, using in vivo and in vitro gain- and loss-of-function approaches, we identified up-regulated DKK1 plays a pivotal role in high-fat diet-induced NAFLD and its progression. Mechanistic analysis reveals that DKK1 enhances the capacity of hepatocytes to uptake fatty acids through the ERK-PPARγ-CD36 axis. Moreover, DKK1 increased insulin resistance by activating the JNK signaling, which in turn exacerbates disorders of hepatic lipid metabolism. Our finding suggests that DKK1 may be a potential therapeutic and diagnosis candidate for NAFLD and metabolic disorder progression.

Modeling MEN1 with Patient-Origin iPSCs Reveals GLP-1R Mediated Hypersecretion of Insulin.

Multiple endocrine neoplasia type 1 (MEN1) is an inherited disease caused by mutations in the MEN1 gene encoding a nuclear protein menin. Among those different endocrine tumors of MEN1, the pancreatic neuroendocrine tumors (PNETs) are life-threatening and frequently implicated. Since there are uncertainties in genotype and phenotype relationship and there are species differences between humans and mice, it is worth it to replenish the mice model with human cell resources. Here, we tested whether the patient-origin induced pluripotent stem cell (iPSC) lines could phenocopy some defects of MEN1. In vitro ß-cell differentiation revealed that the percentage of insulin-positive cells and insulin secretion were increased by at least two-fold in MEN1-iPSC derived cells, which was mainly resulted from significantly higher proliferative activities in the pancreatic progenitor stage (Day 7-13). This scenario was paralleled with increased expressions of prohormone convertase1/3 (PC1/3), glucagon-like peptide-1 (GLP-1), GLP-1R, and factors in the phosphatidylinositol 3-kinase (PI3K)/AKT signal pathway, and the GLP-1R was mainly expressed in ß-like cells. Blockages of either GLP-1R or PI3K significantly reduced the percentages of insulin-positive cells and hypersecretion of insulin in MEN1-derived cells. Furthermore, in transplantation of different stages of MEN1-derived cells into immune-deficient mice, only those ß-like cells produced tumors that mimicked the features of the PNETs from the original patient. To the best of our knowledge, this was the first case using patient-origin iPSCs modeling most phenotypes of MEN1, and the results suggested that GLP-1R may be a potential therapeutic target for MEN1-related hyperinsulinemia.

Efficiently generate functional hepatic cells from human pluripotent stem cells by complete small-molecule strategy.

BACKGROUND: Various methods have been developed to generate hepatic cells from human pluripotent stem cells (hPSCs) that rely on the combined use of multiple expensive growth factors, limiting industrial-scale production and widespread applications. Small molecules offer an attractive alternative to growth factors for producing hepatic cells since they are more economical and relatively stable. METHODS: We dissect small-molecule combinations and identify the ideal cocktails to achieve an optimally efficient and cost-effective strategy for hepatic cells differentiation, expansion, and maturation. RESULTS: We demonstrated that small-molecule cocktail CIP (including CHIR99021, IDE1, and PD0332991) efficiently induced definitive endoderm (DE) formation via increased endogenous TGF-ß/Nodal signaling. Furthermore, we identified that combining Vitamin C, Dihexa, and Forskolin (VDF) could substitute growth factors to induce hepatic specification. The obtained hepatoblasts (HBs) could subsequently expand and mature into functional hepatocyte-like cells (HLCs) by the established chemical formulas. Thus, we established a stepwise strategy with complete small molecules for efficiently producing scalable HBs and functionally matured HLCs. The small-molecule-derived HLCs displayed typical functional characteristics as mature hepatocytes in vitro and repopulating injured liver in vivo. CONCLUSION: Our current small-molecule-based hepatic generation protocol presents an efficient and cost-effective platform for the large-scale production of functional human hepatic cells for cell-based therapy and drug discovery using.

Hypomorphic ASGR1 modulates lipid homeostasis via INSIG1-mediated SREBP signaling suppression.

A population genetic study identified that the asialoglycoprotein receptor 1 (ASGR1) mutation carriers had substantially lower non-HDL-cholesterol (non-HDL-c) levels and reduced risks of cardiovascular diseases. However, the mechanism behind this phenomenon remained unclear. Here, we established Asgr1-knockout mice that represented a plasma lipid profile with significantly lower non-HDL-c and triglyceride (TG) caused by decreased secretion and increased uptake of VLDL/LDL. These 2 phenotypes were linked with the decreased expression of microsomal triglyceride transfer protein and proprotein convertase subtilisin/kexin type 9, 2 key targeted genes of sterol regulatory element-binding proteins (SREBPs). Furthermore, there were fewer nuclear SREBPs (nSREBPs) on account of more SREBPs being trapped in endoplasmic reticulum, which was caused by an increased expression of insulin-induced gene 1 (INSIG1), an anchor of SREBPs. Overexpression and gene knockdown interventions, in different models, were conducted to rescue the ASGR1-deficient phenotypes, and we found that INSIG1 knockdown independently reversed the ASGR1-mutated phenotypes with increased serum total cholesterol, LDL-c, TG, and liver cholesterol content accompanied by restored SREBP signaling. ASGR1 rescue experiments reduced INSIG1 and restored the SREBP network defect as manifested by improved apolipoprotein B secretion and reduced LDL uptake. Our observation demonstrated that increased INSIG1 is a critical factor responsible for ASGR1 deficiency-associated lipid profile changes and nSREBP suppression. This finding of an ASGR1/INSIG1/SREBP axis regulating lipid hemostasis may provide multiple potential targets for lipid-lowering drug development.

Generation of WAe001-A-58 human embryonic stem cell line with inducible expression of the SARS-CoV-2 nucleocapsid protein.

Excessive prostaglandin E2 (PGE2) is the key pathological basis for COVID-19 and a Celebrex treatment of hospitalized COVID-19 patients with comorbidities led to 100% discharged rate and zero death (Hong et al. 2020). It is also suggested that SARS-CoV-2 infected multiple organs and the SARS-CoV nucleocapsid (N) protein transcriptionally drives the expression of the host COX-2 gene. In order to test whether SARS-CoV-2 N protein activates COX-2 transcription in multiple human relevant cell types, an expression inducible human embryonic stem cell line was generated by piggyBac transposon system. This cell line maintained its pluripotency, differentiation potentials, normal morphology and karyotype.

Robust expansion and functional maturation of human hepatoblasts by chemical strategy.

BACKGROUND: Chemically strategies to generate hepatic cells from human pluripotent stem cells (hPSCs) for the potential clinical application have been improved. However, producing high quality and large quantities of hepatic cells remain challenging, especially in terms of step-wise efficacy and cost-effective production requires more improvements. METHODS: Here, we systematically evaluated chemical compounds for hepatoblast (HB) expansion and maturation to establish a robust, cost-effective, and reproducible methodology for self-renewal HBs and functional hepatocyte-like cell (HLC) production. RESULTS: The established chemical cocktail could enable HBs to proliferate nearly 3000 folds within 3 weeks with preserved bipotency. Moreover, those expanded HBs could be further efficiently differentiated into homogenous HLCs which displayed typical morphologic features and functionality as mature hepatocytes including hepatocyte identity marker expression and key functional activities such as cytochrome P450 metabolism activities and urea secretion. Importantly, the transplanted HBs in the injured liver of immune-defect mice differentiated as hepatocytes, engraft, and repopulate in the injured loci of the recipient liver. CONCLUSION: Together, this chemical compound-based HLC generation method presents an efficient and cost-effective platform for the large-scale production of functional human hepatic cells for cell-based therapy and drug discovery application.

Generation of a DKK1 homozygous knockout human embryonic stem cell line WAe001-A-21 using CRISPR/Cas9.

Dickkopf1 (DKK1) is a secreted inhibitor for the Wnt signalling, which is involved in cell proliferation, tissue regeneration and embryonic development. Using CRISPR/Cas9 editing, we established a homozygous mutant DKK1 human embryonic stem cell line (WAe001-A-21). It has a 41 bp deletion in exon 2 of DKK1, leading to its coding frame shift. The WAe001-A-21 cell line maintains a normal karyotype, pluripotency markers, typical stem cell morphology and the ability to differentiate into three germ layers.

Calcitriol alleviates ethanol-induced hepatotoxicity via AMPK/mTOR-mediated autophagy.

Excessive ethanol consumption causes cellular damage, leading to fetal alcohol syndrome and alcohol liver diseases, which are frequently seen with vitamin D (VD) deficiency. A great deal of progress has been achieved in the mechanisms of ethanol-induced hepatocyte damage. However, there are limited intervention means to reduce or rescue hepatocytes damage caused by ethanol. On the basis of our preliminary limited screen process, calcitriol showed a positive effect on protecting hepatocyte viability. Therefore, the molecular basis is worth elucidating. We found that calcitriol pretreatment markedly improved the cell viability, decreased cell apoptosis and oxidative stress and alleviated the abnormal mitochondrial morphology and membrane potential of hepatocytes induced by ethanol. Notably, autophagy was significantly enhanced by calcitriol, as evident by the increasing number of autophagosomes and autolysosomes, upregulated LC3B-Ⅱ and ATG5 levels, and promotion of p62 degradation. Furthermore, calcitriol pretreatment increased the colocalization of GFP-LC3-labeled autophagosomes with mitochondria, suggesting that calcitriol effectively promoted ethanol-induced mitophagy in hepatocytes. In addition, the inhibition of autophagy attenuated the protective and preventive effect of calcitriol. Furthermore, the effect of calcitriol on autophagy was regulated by AMPK/mTOR signaling, and signaling transduction was dependent on the Vitamin D receptor (VDR). In conclusion, calcitriol ameliorates ethanol-induced hepatocyte damage by enhancing autophagy. It may offer a convenient preventive and hepatoprotective mean for people on occasional social drink.

Celebrex Adjuvant Therapy on Coronavirus Disease 2019: An Experimental Study.

Background: The pandemic of coronavirus disease 2019 (COVID-19) resulted in grave morbidity and mortality worldwide. There is currently no effective drug to cure COVID-19. Based on analyses of available data, we deduced that excessive prostaglandin E2 (PGE2) produced by cyclooxygenase-2 was a key pathological event of COVID-19. Methods: A prospective clinical study was conducted in one hospital for COVID-19 treatment with Celebrex to suppress the excessive PGE2 production. A total of 44 COVID-19 cases were enrolled, 37 cases in the experimental group received Celebrex as adjuvant (full dose: 0.2 g, bid; half dose: 0.2 g, qd) for 7-14 days, and the dosage and duration was adjusted for individuals, while seven cases in the control group received the standard therapy. The clinical outcomes were evaluated by measuring the urine PGE2 levels, lab tests, CT scans, vital signs, and other clinical data. The urine PGE2 levels were measured by mass spectrometry. The study was registered and can be accessed at http://www.chictr.org.cn/showproj.aspx?proj=50474. Results: The concentrations of PGE2 in urine samples of COVID-19 patients were significantly higher than those of PGE2 in urine samples of healthy individuals (mean value: 170 ng/ml vs 18.8 ng/ml, p < 0.01) and positively correlated with the progression of COVID-19. Among those 37 experimental cases, there were 10 cases with age over 60 years (27%, 10/37) and 13 cases (35%, 13/37) with preexisting conditions including cancer, atherosclerosis, and diabetes. Twenty-five cases had full dose, 11 cases with half dose of Celebrex, and one case with ibuprofen. The remission rates in midterm were 100%, 82%, and 57% of the full dose, half dose, and control group, respectively, and the discharged rate was 100% at the endpoint with Celebrex treatment. Celebrex significantly reduced the PGE2 levels and promoted recovery of ordinary and severe COVID-19. Furthermore, more complications, severity, and death rate were widely observed and reported in the COVID-19 group of elders and with comorbidities; however, this phenomenon did not appear in this particular Celebrex adjunctive treatment study. Conclusion: This clinical study indicates that Celebrex adjuvant treatment promotes the recovery of all types of COVID-19 and further reduces the mortality rate of elderly and those with comorbidities.

Repair of acute liver damage with immune evasive hESC derived hepato-blasts.

Human embryonic stem cells (hESCs) can undergo unlimited self-renewal and differentiate into hepatic cells, including expandable hepato-blasts (HBs) and hepatocyte-like cells (HLCs) in vitro. Therefore, hESC-derived HBs have the potential to become a renewable cell source for cell therapy of serious liver damage. However, one of the key challenges for such cell therapy is the allogeneic immune rejection of hESC-derived HBs. To overcome this challenge, we developed a strategy to protect the hESC-derived HBs from allogeneic immune rejection by ectopically expressing immune suppressive molecules CTLA4-Ig and PD-L1, denoted CP HBs. Like HBs derived from normal hESCs, CP HBs are capable of repairing liver damage in animal models. Using humanized mice (Hu-mice) reconstituted with human immune system, we showed that CP HBs are protected from allogeneic immune system and can survive long-term in Hu-mice. These data support the feasibility to develop CP HBs into a cell therapy to treat serious liver damage.

Kupffer cells mediate the recruitment of hepatic stellate cells into the localized liver damage.

Currently, there is a growing interest in understanding the cellular and molecular events of immune-cell trafficking and recruitment of hepatic stellate cells (HSCs) in liver diseases. Aberrant activation of HSCs is the key event leading to chronic liver fibrosis. However, the underlying mechanisms of the recruitment of HSCs in a locally injured liver are not clearly understood. Here, we report a new experimental approach for the study of inflammatory responses as well as the recruitment of HSCs into the localized cryolesion. We observed a significant liver damage accompanied by the up-regulation of plasma ALT and AST. In addition, we also found increased levels of MCP-1, IL-6 and IL-10 cytokines. The peak cytokine levels were detected at 8 h after injury, followed by intrahepatic infiltration of neutrophils and monocytes into the injury site (from 8 h to day 3), while the kupffer cells (KCs) and HSCs were mainly detected on day 3 after injury. Interestingly, the depletion of KCs, but not neutrophils, reduced the directional recruitment and accumulation of HSCs at the injury site. Moreover, the combinatorial recruitment of KCs and HSCs resulted in the gradual restoration of fibrotic area to almost typical histological appearance on day 14 post-injury. In conclusion, our data demonstrated a localized infiltration and accumulation of neutrophils and monocytes at a "predefined loci", and further revealed that KCs are critical for the recruitment of HSCs during injury, and thus, may play an important role in tissue repair.

Ascorbate protects liver from metabolic disorder through inhibition of lipogenesis and suppressor of cytokine signaling 3 (SOCS3).

BACKGROUND: Fatty liver is a reversible status, but also an origin stage to develop to other metabolic syndromes, such as diabetes and heart disease that threatens public health worldwide. Ascorbate deficiency is reported to be correlated with increasing risks for metabolic syndromes, but whether ascorbate has a therapeutic effect is unknown. Here, we investigated if ascorbate treatment alone could work on protecting from the development of steatosis and mechanisms beyond. METHODS: Guinea pigs were fed with a chow diet or a high palm oil diet (HPD) respectively. HPD induced animals were administered different concentrations of ascorbate in different time intervals through water. Besides, hepatocyte-like cells derived from human embryonic stem cells and HepG2 cells were treated with palmitic acid (PA) to induce lipid accumulation for molecular mechanism study. RESULTS: We find that ascorbate rescues HPD and PA induced steatosis and insulin tolerance in vivo and in vitro. We demonstrate that ascorbate changes cellular lipid profiles via inhibits lipogenesis, and inhibits the expression of SOCS3 via STAT3, thus enhances insulin signal transduction. Overexpression of SOCS3 abolishes the ascorbate rescue effects on insulin signal and lipid accumulation in hepatic cells. CONCLUSIONS: Ascorbate ameliorates hepatic steatosis and improves insulin sensitivity through inhibiting lipogenesis and SOCS3.

The correlation and role analysis of COL4A1 and COL4A2 in hepatocarcinogenesis.

Liver fibrosis biomarker, Type IV collagen, may function as hepatocarcinogenesis niche. However, among the six isoforms, the isoforms providing tumor microenvironment and their regulatory network are still unclarified. Based on bioinformatics analysis of hundreds of HCC transcriptome datasets from public databases, we found that COL4A1/2 expressions were significantly correlated with hepatocarcinogenesis, progression, and prognosis. The expressions of COL4A1/2 were significantly upregulated in the preneoplastic and HCC tissues compared with normal tissues. Moreover, the overexpression of COL4A2 was highly correlated with shorter progression-free survival in HCC patients. Bioinformatics analysis also generates an interactive regulatory network in which COL4A1/2 directly binding to integrin alpha-2/beta-1 initiates a sequentially and complicated signaling transduction, to accelerate cell cycle and promote tumorigenesis. Among those pathways, the PI3K-Akt pathway is significantly enriched in cooperative mutations and correlation analysis. This suggests that the key activated signaling is PI3K-Akt pathway which severing as the centerline linked with other pathways (Wnt and MAPK signaling) and cell behaviors signaling (cell cycle control and cytoskeleton change). Switching extracellular matrix collagen isoform may establish pro-tumorigenic and metastatic niches. The findings of COL4A1/2 and related signaling networks are valuable to be further investigated that may provide druggable targets for HCC intervention.

Synergistic modulation of signaling pathways to expand and maintain the bipotency of human hepatoblasts.

BACKGROUND: The limited proliferative ability of hepatocytes is a major limitation to meet their demand for cell-based therapy, bio-artificial liver device, and drug tests. One strategy is to amplify cells at the hepatoblast (HB) stage. However, expansion of HBs with their bipotency preserved is challenging. Most HB expansion methods hardly maintain the bipotency and also lack functional confirmation. METHODS: On the basis of analyzing and manipulating related signaling pathways during HB (derived from human induced pluripotent stem cells, iPSCs) differentiation and proliferation, we established a specific chemically defined cocktails to synergistically regulate the related signaling pathways that optimize the balance of HB proliferation ability and stemness maintenance, to expand the HBs and investigate their capacity for injured liver repopulation in immune-deficient mice. RESULTS: We found that the proliferative ability progressively declines during HB differentiation process. Small molecule activation of Wnt or inhibition of TGF-ß pathways promoted HB proliferation but diminished their bipotency, whereas activation of hedgehog (HH) signaling stimulated proliferation and sustained HB phenotypes. A cocktail synergistically regulating the BMP/WNT/TGF-ß/HH pathways created a fine balance for expansion and maintenance of the bipotency of HBs. After purification, colony formation, and expansion for 20 passages, HBs retained their RNA profile integrity, normal karyotype, and ability to differentiate into mature hepatocytes and cholangiocytes. Moreover, upon transplantation into liver injured mice, the expanded HBs could engraft and differentiate into mature human hepatocytes and repopulate liver tissue with restoring hepatocyte mass. CONCLUSION: Our data contribute to the understanding of some signaling pathways for human HB proliferation in vitro. Simultaneous BMP/HGF induction, activation of Wnt and HH, and inhibition of TGF-ß pathways created a reliable method for long-term stable large-scale expansion of HBs to obtain mature hepatocytes that may have substantial clinical applications.

Direct Conversion of Human Urine Cells to Neurons by Small Molecules.

Transdifferentiation of other cell type into human neuronal cells (hNCs) provides a platform for neural disease modeling, drug screening and potential cell-based therapies. Among all of the cell donor sources, human urine cells (hUCs) are convenient to obtain without invasive harvest procedure. Here, we report a novel approach for the transdifferentiation of hUCs into hNCs. Our study demonstrated that a combination of seven small molecules (CAYTFVB) cocktail induced transdifferentiation of hUCs into hNCs. These chemical-induced neuronal cells (CiNCs) exhibited typical neuron-like morphology and expressed mature neuronal markers. The neuronal-like morphology revealed in day 1, and the Tuj1-positive CiNCs reached to about 58% in day 5 and 38.36% Tuj1+/MAP2+ double positive cells in day 12. Partial electrophysiological properties of CiNCs was obtained using patch clamp. Most of the CiNCs generated using our protocol were glutamatergic neuron populations, whereas motor neurons, GABAergic or dopaminergic neurons were merely detected. hUCs derived from different donors were converted into CiNCs in this work. This method may provide a feasible and noninvasive approach for reprogramming hNCs from hUCs for disease models and drug screening.

Construction of a GLI3 compound heterozygous knockout human embryonic stem cell line WAe001-A-20 by CRISPR/Cas9 editing.

The human GLI3 protein has a dual function as a transcriptional activator or repressor of hedgehog signaling, depending on the proteolytic processing forms of GLI3. In this study, we established a compound heterozygous GLI3 mutant human embryonic stem cell line (WAe001-A-20) through CRISPR/Cas9 editing. The WAe001-A-20 cells carried two deletions on two different alleles of exon 2 of GLI3, respectively, which resulted in a frame shift and early termination in the translation of GLI3. Moreover, WAe001-A-20 maintains a normal karyotype, parental cell morphology, pluripotent phenotype and the ability to differentiate into three germ layers. Resource table.

New Turns for High Efficiency Knock-In of Large DNA in Human Pluripotent Stem Cells.

The groundbreaking CRISPR technology is revolutionizing biomedical research with its superior simplicity, high efficiency, and robust accuracy. Recent technological advances by a coupling CRISPR system with various DNA repair mechanisms have further opened up new opportunities to overcome existing challenges in knocking-in foreign DNA in human pluripotent stem cells, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). In this review, we summarized the very recent development of CRISPR-based knock-in strategies and discussed the results obtained as well as potential applications in human ESC and iPSC.

Generation of WAe001-A-15, a human embryonic stem cell line with miR-122 doxycycline-inducible expression.

MiR-122 is the most abundant miRNA in the human liver accounting for 52% of the entire hepatic miRNome. Previous studies have demonstrated that miR-122 is a valuable therapeutic target for liver diseases, including viral hepatitis, fibrosis, steatosis, and hepatocarcinoma. Here, we constructed a miR-122 doxycycline-inducible expression human embryonic stem cell line WAe001-A-15 using the piggyBac transposon system. The cell line retained its pluripotency, in vitro differentiation potential, normal morphology, and karyotype.

Generation of a GDE heterozygous mutation human embryonic stem cell line WAe001-A-14 by CRISPR/Cas9 editing.

Glycogen debranching enzyme (GDE) plays a critical role in glycogenolysis. Mutations in the GDE gene are associated with a metabolic disease known as glycogen storage disease type III (GSDIII). We generated a mutant GDE human embryonic stem cell line, WAe001-A-14, using the CRISPR/Cas9 editing system. This cell line contains a 24-nucleotide deletion within exon-13 of GDE, resulting in 8 amino acids (TRLGISSL) missing of the GDE protein from amino acid position 567 to 575. The WAe001-A-14 cell line maintains typical stem cell morphology, pluripotency and in vitro differentiation potential, and a normal karyotype.