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Currently, there is no effective treatment for obesity and alcohol-associated liver diseases, partially due to the lack of translational human models. Here, we present a protocol to generate 3D human liver spheroids that contain all the liver cell types and mimic "livers in a dish." We describe strategies to induce metabolic and alcohol-associated hepatic steatosis, inflammation, and fibrosis. We outline potential applications, including using human liver spheroids for experimental and translational research and drug screening to identify potential anti-fibrotic therapies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Liver fibrosis of different etiologies is a serious health problem worldwide. There is no effective therapy available for liver fibrosis except the removal of the underlying cause of injury or liver transplantation. Development of liver fibrosis is caused by fibrogenic myofibroblasts that are not present in the normal liver, but rather activate from liver resident mesenchymal cells in response to chronic toxic or cholestatic injury. Many studies indicate that liver fibrosis is reversible when the causative agent is removed. Regression of liver fibrosis is associated with the disappearance of activated myofibroblasts and resorption of the fibrous scar. In this review, we discuss the results of genetic tracing and cell fate mapping of hepatic stellate cells and portal fibroblasts, their specific characteristics, and potential phenotypes. We summarize research progress in the understanding of the molecular mechanisms underlying the development and reversibility of liver fibrosis, including activation, apoptosis, and inactivation of myofibroblasts., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Sterol regulatory element-binding proteins (SREBPs) are master transcription factors that play a crucial role in regulating genes involved in the biogenesis of cholesterol, fatty acids, and triglycerides. As such, they are implicated in several serious liver diseases, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). SREBPs are subject to regulation by multiple cofactors and critical signaling pathways, making them an important target for therapeutic interventions. In this review, we first introduce the structure and activation of SREBPs, before focusing on their function in liver disease. We examine the mechanisms by which SREBPs regulate lipogenesis, explore how alterations in these processes are associated with liver disease, and evaluate potential therapeutic strategies using small molecules, natural products, or herb extracts that target these pathways. Through this analysis, we provide new insights into the versatility and multitargets of SREBPs as factors in the modulation of different physiological stages of liver disease, highlighting their potential targets for therapeutic treatment.

Here, we present a protocol for isolating human hepatocytes and neural progenitor cells from normal and nonalcoholic steatohepatitis livers. We describe steps for perfusion for scaled-up liver cell isolation and optimization of chemical digestion to achieve maximal yield and cell viability. We then detail a liver cell cryopreservation and potential applications, such as the use of human liver cells as a tool to link experimental and translational research., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

With an increasing number of patients with degenerative hepatic diseases, such as liver fibrosis, and a limited supply of donor organs, there is an unmet need for therapies that can repair or regenerate damaged liver tissue. Treatment with macrophages that are capable of phagocytosis and anti-inflammatory activities such as secretion of matrix metalloproteinases (MMPs) provide an attractive cellular therapy approach. Human induced pluripotent stem cells (iPSCs) are capable of efficiently generating a large-scale, homogenous population of human macrophages using fully defined feeder- and serum-free differentiation protocol. Human iPSC-macrophages exhibit classical surface cell markers and phagocytic activity similar to peripheral blood-derived macrophages. Moreover, gene and cytokine expression analysis reveal that these macrophages can be efficiently polarized to pro-inflammatory M1 or anti-inflammatory M2 phenotypes in presence of LPS + IFN-γ and IL-4 + IL-13, respectively. M1 macrophages express high level of CD80, TNF-α, and IL-6 while M2 macrophages show elevated expression of CD206, CCL17, and CCL22. Here, we demonstrate that treatment of liver fibrosis with both human iPSC-derived macrophage populations and especially M2 subtype significantly reduces fibrogenic gene expression and disease associated histological markers including Sirius Red, αSMA and desmin in immunodeficient Rag2 -/- γc -/- mice model, making this approach a promising cell-based avenue to ameliorate fibrosis., (© 2021 AlphaMed Press.)

Alcoholic liver disease (ALD) is a leading cause of cirrhosis in the United States, which is characterized by extensive deposition of extracellular matrix proteins and formation of a fibrous scar. Hepatic stellate cells (HSCs) are the major source of collagen type 1 producing myofibroblasts in ALD fibrosis. However, the mechanism of alcohol-induced activation of human and mouse HSCs is not fully understood. We compared the gene-expression profiles of primary cultured human HSCs (hHSCs) isolated from patients with ALD (n = 3) or without underlying liver disease (n = 4) using RNA-sequencing analysis. Furthermore, the gene-expression profile of ALD hHSCs was compared with that of alcohol-activated mHSCs (isolated from intragastric alcohol-fed mice) or CCl 4 -activated mouse HSCs (mHSCs). Comparative transcriptome analysis revealed that ALD hHSCs, in addition to alcohol-activated and CCl 4 -activated mHSCs, share the expression of common HSC activation ( Col1a1 [collagen type I alpha 1 chain], Acta1 [actin alpha 1, skeletal muscle], PAI1 [plasminogen activator inhibitor-1], TIMP1 [tissue inhibitor of metalloproteinase 1], and LOXL2 [lysyl oxidase homolog 2]), indicating that a common mechanism underlies the activation of human and mouse HSCs. Furthermore, alcohol-activated mHSCs most closely recapitulate the gene-expression profile of ALD hHSCs. We identified the genes that are similarly and uniquely up-regulated in primary cultured alcohol-activated hHSCs and freshly isolated mHSCs, which include CSF1R (macrophage colony-stimulating factor 1 receptor), PLEK (pleckstrin), LAPTM5 (lysosmal-associated transmembrane protein 5), CD74 (class I transactivator, the invariant chain), CD53 , MMP9 (matrix metallopeptidase 9), CD14 , CTSS (cathepsin S), TYROBP (TYRO protein tyrosine kinase-binding protein), and ITGB2 (integrin beta-2), and other genes (compared with CCl 4 -activated mHSCs). Conclusion: We identified genes in alcohol-activated mHSCs from intragastric alcohol-fed mice that are largely consistent with the gene-expression profile of primary cultured hHSCs from patients with ALD. These genes are unique to alcohol-induced HSC activation in two species, and therefore may become targets or readout for antifibrotic therapy in experimental models of ALD., (© 2020 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Aim: To investigate the therapeutic effect of FZHY on hepatic fibrosis in mice and to determine the mechanism of its action., Methods: Wild type mice were subjected to toxic (carbon tetrachloride, CCl 4 ) or cholestatic (bile duct ligation, BDL). Upon induction of liver fibrosis, mice were treated with FZHY (4.0g/kg, 2w, oral gavage) or vehicle (PBS). Livers were analyzed by Sirius Red staining, immunostaining and RT-PCR for profibrogenic and pro-inflammatory genes. The effect of FZHY on hepatocytes, inflammatory responses, activation of fibrogenic myofibroblasts, and ROS production was assessed., Results: FZHY strongly inhibited the development of CCl 4 - and BDL-induced liver fibrosis in mice. Liver fibrosis was significantly improved in FZHY-treated mice, as demonstrated by reduced content of hepatic hydroxyproline and Sirius Red positive area. Moreover, the number of SMA + and Desmin + myofibroblasts was significantly reduced in the livers of FZHY-treated mice, and correlated with downregulation of the mRNA levels of α-SMA, collagen-α1(I), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), TGF-β1 and its receptor TGF-βRI, and platelet-derived growth factor-β (PDGF-β), suggesting that FZHY inhibits activation of fibrogenic myofibroblasts. Furthermore, administration of FZHY markedly decreased recruitment of F4/80 + inflammatory macrophages to the livers of CCl 4 - and BDL-injured mice, and this effect was associated with downregulation of monocyte chemoattractant protein-1(MCP-1) and macrophage inflammatory protein-1 (MIP-1) mRNA. In addition, the lipid peroxidation products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) were reduced, demonstrating that treatment with FZHY can effectively block ROS production in livers of CCl 4 - and BDL-injured mice., Conclusions: Traditional Chinese Medicine FZHY has a variety of anti-fibrotic effects, including strong anti-oxidant, anti-inflammatory and anti-fibrotic effects on myeloid cells and hepatocytes. Although FZHY compound does not seem to directly affect HSCs, it regulates HSC activation via inhibition of macrophage recruitment to fibrotic liver.

We aimed to investigate whether aging increases the susceptibility of hepatic and renal inflammation or fibrosis in response to high-fat diet (HFD) and explore the underlying genetic alterations. Middle (10 months old) and old (20 months old) aged, male C57BL/6N mice were fed either a low-fat diet (4 % fat) or HFD (60 % fat) for 4 months. Young (3 months old) aged mice were included as control group. HFD-induced liver and kidney injuries were analyzed by serum and urine assay, histologic staining, immunohistochemistry, and reverse-transcription real-time quantitative polymerase chain reaction. Total RNA sequencing with next-generation technology was done with RNA extracted from liver tissues. With HFD feeding, aged was associated with higher serum alanine aminotransferase levels, marked infiltration of hepatic macrophages, and increased expression of inflammatory cytokines (MCP1, TNF-α, IL-1β, IL-6, IL-12, IL-17A). Importantly, aged mice showed more advanced hepatic fibrosis and increased expression of fibrogenic markers (Col-I-α1, αSMA, TGF-β1, TGF-β2, TGFβRII, PDGF, PDGFRβII, TIMP1) in response to HFD. Aged mice fed on HFD also showed increased oxidative stress and TLR4 expression. In the total RNA seq and gene ontology analysis of liver, old-aged HFD group showed significant up-regulation of genes linked to innate immune response, immune response, defense response, inflammatory response compared to middle-aged HFD group. Meanwhile, aging and HFD feeding showed significant increase in glomerular size and mesangial area, higher urine albumin/creatinine ratio, and advanced renal inflammation or fibrosis. However, the difference of HFD-induced renal injury between old-aged group and middle-aged group was not significant. The susceptibility of hepatic fibrosis as well as hepatic inflammation in response to HFD was significantly increased with aging. In addition, aging was associated with glomerular alterations and increased renal inflammation or fibrosis, while the differential effect of aging on HFD-induced renal injury was not remarkable as shown in the liver., Competing Interests: None. Financial support and sponsorship This research was supported by The GlaxoSmithKline Research Fund of the Korean Association for the study of the Liver (In Hee Kim). Support: NIH R01MH094151-01 and MH019934, and the Sam and Rose Stein Institute for Research on Aging, University of California, San Diego.

Physical mapping of human chromosomes at a resolution of 100 kb to 1 Mb will provide important reagents for gene identification and framework templates for ultimately determining the complete DNA sequence. Sequence-tagged site (STS) content mapping, coupled with large fragment cloning in yeast artificial chromosomes, provides an efficient mechanism for producing first-generation, low-resolution maps of human chromosomes. Previously, we produced a set of standardized STSs for human chromosome 11 regionally localized by fluorescence in situ hybridization or somatic cell hybrid analysis. In this paper, we used these as well as other STS content, and identify 109 islands spanning an estimated 218 Mb on the 126-Mb chromosome. Since about 62% of the islands contain markers ordered on chromosome 11 by genetic or radiation hybrid analysis, this data set represents a first-order approximation of a physical map of human chromosome 11. This set of clones, contigs, and associated STSs will provide the material for the production of a continuous overlapping set of YACs as well for high-resolution physical mapping based upon sampled and complete DNA sequencing.

We report the construction of 370 sequence-tagged sites (STSs) that are detectable by PCR amplification under sets of standardized conditions and that have been regionally mapped to human chromosome 11. DNA sequences were determined by sequencing directly from cosmid templates using primers complementary to T3 and T7 promoters present in the cloning vector. Oligonucleotide PCR primers were predicted by computer and tested using a battery of genomic DNAs. Cosmids were regionally localized on chromosome 11 by using fluorescence in situ hybridization or by analyzing a somatic cell hybrid panel. Additional STSs corresponding to known genes and markers on chromosome 11 were also produced under the same series of standardized conditions. The resulting STSs provide uniform coverage of chromosome 11 with an average spacing of 340 kb. The DNA sequence determined for use in STS production corresponds to about 0.1% (116 kb) of chromosome 11 and has been analyzed for the presence of repetitive sequences, similarities to known genes and motifs, and possible exons. Computer analysis of this sequence has identified and therefore mapped at least eight new genes on chromosome 11.