Kupffer cells abrogate homing and repopulation of allogeneic hepatic progenitors in injured liver site.

BACKGROUND: Allogeneic hepatocyte transplantation is an emerging approach to treat acute liver defects. However, durable engraftment of the transplanted cells remains a daunting task, as they are actively cleared by the recipient's immune system. Therefore, a detailed understanding of the innate or adaptive immune cells-derived responses against allogeneic transplanted hepatic cells is the key to rationalize cell-based therapies. METHODS: Here, we induced an acute inflammatory regenerative niche (3-96 h) on the surface of the liver by the application of cryo-injury (CI) to systematically evaluate the innate immune response against transplanted allogeneic hepatic progenitors in a sustained micro-inflammatory environment. RESULTS: The resulting data highlighted that the injured site was significantly repopulated by alternating numbers of innate immune cells, including neutrophils, monocytes and Kupffer cells (KCs), from 3 to 96 h. The transplanted allo-HPs, engrafted 6 h post-injury, were collectively eliminated by the innate immune response within 24 h of transplantation. Selective depletion of the KCs demonstrated a delayed recruitment of monocytes from day 2 to day 6. In addition, the intrasplenic engraftment of the hepatic progenitors 54 h post-transplantation was dismantled by KCs, while a time-dependent better survival and translocation of the transplanted cells into the injured site could be observed in samples devoid of KCs. CONCLUSION: Overall, this study provides evidence that KCs ablation enables a better survival and integration of allo-HPs in a sustained liver inflammatory environment, having implications for rationalizing the cell-based therapeutic interventions against liver defects.

Neutralizing serum amyloid a protects against sinusoidal endothelial cell damage and platelet aggregation during acetaminophen-induced liver injury.

Serum amyloid A (SAA) is an acute response protein that mainly produced by hepatocytes, and it can promote endothelial dysfunction via a pro-inflammatory and pro-thrombotic effect in atherosclerosis and renal disease. Overdose of Acetaminophen (APAP) will cause hepatotoxicity accompany with hepatocyte necrosis, liver sinusoidal endothelial cells (LSECs) damage and thrombosis in liver. However, whether SAA plays a role in APAP-induced liver toxicity remains unclear. Here, we evaluated the Saa1/2 expression in APAP-induced liver injury, and found that Saa1/2 production was significantly increased in an autocrine manner in APAP injury model. Moreover, we used neutralizing antibody (anti-SAA) to block the function of serum Saa1/2. We found that neutralizing serum Saa1/2 protected against APAP-induced liver injuries and increased the survival rate of mice that were treated with lethal dose APAP. Further investigations showed that blocking Saa1/2 reduced APAP-induced sinusoidal endothelium damage, hemorrhage and thrombosis. In addition, in vitro experiments showed that Saa1/2 augmented the toxic effect of APAP on LSECs, and Saa1/2 promoted platelets aggregation on LSECs cell membrane. Taken together, this study suggests that Saa1/2 may play a critical role in APAP-induced liver damages through platelets aggregation and sinusoidal damage. Therefore, we conceptually demonstrate that inhibition of SAA may be a potential intervention for APAP-directed acute liver injuries.

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.

Inhibition of Hedgehog Delays Liver Regeneration through Disrupting the Cell Cycle.

Liver regeneration is a complicated biological process orchestrated by various liver resident cells. Hepatic cell proliferation and reconstruction of the hepatic architecture involve multiple signaling pathways. It has been reported that the Hh signal is involved in liver regeneration. However, the signal transduction pathways and cell types involved are ill studied. This study aimed to investigate hedgehog signal response cell types and the specific molecular mechanism involved in the process of liver regeneration. Partial hepatectomy (PH) of 70% was performed on ICR (Institute of Cancer Research) mice to study the process of liver regeneration. We found that the hedgehog signal was activated significantly after PH, including hedgehog ligands, receptors and intracellular signaling molecules. Ligand signals were mainly expressed in bile duct cells and non-parenchymal hepatic cells, while receptors were expressed in hepatocytes and some non-parenchymal cells. Inhibition of the hedgehog signal treated with vismodegib reduced the liver regeneration rate after partial hepatectomy, including inhibition of hepatic cell proliferation by decreasing Cyclin D expression and disturbing the cell cycle through the accumulation of Cyclin B. The current study reveals the important role of the hedgehog signal and its participation in the regulation of hepatic cell proliferation and the cell cycle during liver regeneration. It provides new insight into the recovery of the liver after liver resection.

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.

SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury.

Hepatic stellate cells (HSCs) are crucial for liver injury repair and cirrhosis. However, the mechanism of chemotactic recruitment of HSCs into injury loci is still largely unknown. Here, we demonstrate that serum amyloid A1 (SAA1) acts as a chemokine recruiting HSCs toward injury loci signaling via TLR2, a finding proven by gene manipulation studies in cell and mice models. The mechanistic investigations revealed that SAA1/TLR2 axis stimulates the Rac GTPases through PI3K-dependent pathways and induces phosphorylation of MLC (pSer19). Genetic deletion of TLR2 and pharmacological inhibition of PI3K diminished the phosphorylation of MLCpSer19 and migration of HSCs. In brief, SAA1 serves as a hepatic endogenous chemokine for the TLR2 receptor on HSCs, thereby initiating PI3K-dependent signaling and its effector, Rac GTPases, which consequently regulates actin filament remodeling and cell directional migration. Our findings provide novel targets for anti-fibrosis drug development.

Calcitriol promotes the maturation of hepatocyte-like cells derived from human pluripotent stem cells.

Human hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) represent a promising cell source for the assessment of hepatotoxicity and pharmaceutical safety testing. However, the hepatic functionality of HLCs remains significantly inferior to primary human hepatocytes. The bioactive vitamin D (VD), calcitriol, promotes the differentiation of many types of cells, and its deficiency is correlated to the severity of liver diseases. Whether calcitriol contributes to the differentiation of HLCs needs to be explored. Here, we found that the supplementation of calcitriol improved the functionalities of hPSCs-derived HLCs in P450 activities, urea production, and albumin secretion. Moreover, calcitriol also enhanced mitochondrial respiratory function with increased protein expression levels of the subunit of respiratory enzyme complexes in HLCs. Further analyses showed that the mitochondrial biogenesis regulators and mitophagy were increased by calcitriol, thus improving the mitochondrial quality. These improvements in functionality and mitochondrial condition were dependent on vitamin D receptor (VDR) because the improvements were abolished under VDR-deficient conditions. Our finding provides a cost-effective chemical process for HLC maturation to meet the demand for basic research and potential clinic applications.

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.

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.

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.

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.

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.

Generation of a SMO homozygous knockout human embryonic stem cell line WAe001-A-16 by CRISPR/Cas9 editing.

The human SMO protein encoded by the smoothened (SMO) gene acts as a positive mediator for Hedgehog signaling. This pathway regulates many cellular activities, developmental morphogenesis, and tumorigenesis. Using CRISPR/Cas9 to edit human embryonic stem cell line WA01 (H1), we established a SMO mutant cell line (WAe001-A-16). This cell line has a 40bp homozygous deletion in exon 2 of SMO leading to a shift in the open reading frame and early termination at amino acid position 287. WAe001-A-16 maintains a normal karyotype, parental cell morphology, pluripotency markers, and the capacity to differentiate into all three germline layers.

Generation of three miR-122 knockout lines from a human embryonic stem cell line.

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 plays key roles in hepatocyte growth, metabolism, and homeostasis. Here, we created three miR-122 knockout human embryonic stem cell line lines, WAe001-A-7, WAe001-A-8, and WAe001-A-9, using the CRISPR/Cas9 technique. These mutated cell lines retained their pluripotency, in vitro differentiation potential, normal morphology, and karyotype.

Testosterone improves the differentiation efficiency of insulin-producing cells from human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) may provide potential resource for regenerative medicine research, including generation of insulin-producing cells for diabetes research and insulin production. Testosterone (T) is an androgen hormone which promotes protein synthesis and improves the management of type 2 diabetes in clinical studies. Concurrently, co-existed hyperandrogenism and hyperinsulinism is frequently observed in polycystic ovary syndrome, congenital adrenal hyperplasia and some of Wermer's syndrome. However, the relationship among androgens, insulin and the differentiation of pancreatic ß cells is still not fully clear. Here we find that T improves the differentiation efficiency of insulin-producing cells from hiPSCs. The addition of T into routine differentiation formula for pancreatic ß cells increases the differentiation efficiency from 12% to 35%. The administration of T promotes the expression of key genes associated with ß cells differentiation including NGN3, NEUROD1 and INS. This finding benefits the ongoing process to optimize the differentiation protocol of pancreatic ß cells from hiPSCs, and provides some degree of understanding the clinical management of T for type 2 diabetes.

[Berberine activates volume-sensitive chloride channel in human colorectal carcinoma cells].

The present study aimed to clarify the effect of berberine on the chloride channels in human colorectal carcinoma cells (SW480). The whole-cell patch clamp technique was used to detect the Cl(-) current activated by berberine. The physiological and pharmacological characteristics of the current were clarified by changing the osmotic pressure of extracellular perfusate and applying chloride channel blockers. The results showed that, under isotonic conditions, the background current of SW480 cells was weak and stable. A large current was induced by perfusing the cells with the isotonic solution containing berberine (10 nmol/L), current density being (85.8 ± 4.6) pA/pF at +80 mV, (-71.9 ± 3.5) pA/pF at -80 mV, with a latency of (115.6 ± 21.7) s. The chloride current showed weak outward rectification and negligible time- and voltage-dependent inactivation. The reversal potential (-5.5 mV ± 1.2 mV) of the current was close to the calculated equilibrium potential for Cl(-) (ECl = -0.9 mV). Experiments under different osmotic pressures showed that the properties of hypotonicity-activated current recorded in SW480 cells were similar to those of the current induced by berberine, and hypertonic solutions suppressed the berberine-induced current by (98.6 ± 2.3)%. On the other hand, berberine-induced Cl(-) current was significantly inhibited by the chloride channel blockers NPPB (100 µmol/L) and tamoxifen (20 µmol/L), with the inhibition ratios of (83.1 ± 3.6)% and (95.6 ± 1.2)% respectively. These results suggest that berberine can activate the chloride channels that are sensitive to NPPB and tamoxifen, as well as the changes of cell volume in human colorectal carcinoma cells.