Intracellular membraneless organelles, including granules, bodies, speckles, etc., play critical roles in physiological and pathological processes. The discovery of new membraneless organelles has generated significant attention. DEAD-box helicase (DDX) family members possess the potential to undergo liquid-liquid phase separation (LLPS), the foundation for the assembly of membraneless organelles. Here, to identify new granules assembled in steatotic hepatocytes, we screened DDX family members and found that lipids, especially arachidonic acid (AA) metabolites, induced LLPS of DDX49 in hepatocytes, forming an assembled granule named as lipid-induced granule (LIG). The assembled LIGs by DDX49 feedback restrained metabolic dysfunction-associated steatotic liver disease (MASLD)-associated fibrosis. Mechanistically, C5-methylcytosine (m5C)-modified mRNA of pro-fibrotic hepatokine tissue inhibitor of metalloproteinase 2 (Timp2) and its reader Y-box binding protein 1 (YBX1) were recruited into LIGs, thereby inhibiting Timp2 mRNA translation and consequently feedback suppressing liver fibrosis. Moreover, LIGs were identified in human MASLD livers and exhibited reverse correlation with fibrosis progression. Therefore, we identified a new granule in steatotic hepatocytes and elucidated its role in restraining liver fibrosis.

Metabolic dysfunction-associated steatotic liver disease (MASLD), especially its severe form, metabolic dysfunction-associated steatohepatitis (MASH), progresses to liver fibrosis, leading to cirrhosis and liver cancer. The cross-talk between spleen and liver in MASLD/MASH progression remains poorly understood. Here we found enlarged spleens in patients with MASLD and identified induced TRNP1hiCD8+ T cells in spleens of MASLD/MASH mouse models and patients. These cells exhibited pro-fibrotic properties through secretion of INSR-α. Mechanistically, demethylated DNA and H3K27me3, increased H3K27ac and bolstered enhancer-promoter contact synergistically reorganized chromatin topologically associating domains spatially to initiate the expression of transcription factor TRNP1 in splenic CD8+ T cells. TRNP1 then transcriptionally activated the expression of FURIN and CTSD, promoting the maturation and ectodomain shedding of INSR-α and facilitating its secretion to activate hepatic stellate cells. In vivo blockade of INSR-α using neutralizing antibodies alleviated MASLD/MASH-induced liver fibrosis. This study reveals splenic TRNP1hiCD8+ T cells with pro-fibrotic properties and suggests a potential anti-fibrotic strategy.

BACKGROUND & AIMS:Liver fibrosis, a common outcome of chronic liver diseases, is generally considered irreversible. Upregulation of clusterin (CLU), a secreted glycoprotein, occurs in several degenerative diseases and is suggested to have protective effects; however, its potential in treating or reversing liver fibrosis requires investigation. METHODS:Induction of CLU and its receptor low-density lipoprotein receptor-related protein 2 (LRP2) in fibrotic liver were determined using single-cell RNA sequencing and immunofluorescence. Its role and mechanism were examined in CLU knockout mice and mesothelial-specific LRP2 knockout mice. A peptide mimicking CLU was designed and verified. RESULTS:CLU expression was induced in pericentral senescent hepatocytes and then secreted during liver fibrosis in both patients and mice. CLU deletion exacerbated liver fibrosis, whereas treatment with recombinant CLU reduced fibrosis, suggesting that CLU induction feedback inhibits fibrosis progression. Mechanistically, LRP2, the receptor for CLU, is expressed specifically by liver mesothelial cells but not hepatic stellate cells. Recognition of CLU by LRP2 triggers mesothelial cell proliferation and migration from the liver surface into the pericentral region, followed by endocytosis of surrounding fibers, thus reducing liver fibrosis. Thereafter, we designed a 20-amino acid peptide mimicking CLU, named CLUtide, and determined its effect in resolving liver fibrosis, suggesting the considerable therapeutic potential. CONCLUSIONS:Pericentral senescent hepatocytes secrete CLU to induce the proliferation and migration of mesothelial cells to endocytose fibers by its receptor LRP2, thus reducing liver fibrosis. The engineered CLUtide mimicking CLU may be promising for resolving hepatic deposited fibers during liver fibrosis.

Manufacturing sufficient quantities of high-quality hepatocytes holds significant promise for the treatment of liver diseases and drug screening. Here, we developed a chemically defined, animal-free method for the large-scale production of human gallbladder epithelial cells (hGBECs) under good manufacturing practice conditions, enabling their clinical application. The cell products were characterized for growth ability, phenotype, freeze-thaw viability, genetic stability, biological contamination, tumorigenicity, and acute toxicity to ensure quality control and biological safety. We also provide a protocol for generating functional hepatocytes from hGBECs. The derived hepatocytes demonstrated typical liver functions, including albumin secretion, urea production, and drug metabolism. In addition, these cells were used in drug toxicity testing. We conducted further functional experiments on Cu2+ transport and alcohol metabolism. Transplantation of these cells in vivo was able to rescue mice from liver failure. This large-scale, convenient strategy for manufacturing hGBECs serves as a biobank for clinical applications and provides a valuable model for studying liver diseases.

The large-scale production of functional hepatocytes is critical for liver disease treatment, disease modeling, and drug development. Here, we present a protocol for isolating, expanding, and maintaining human gallbladder epithelial cells (hGBECs). We describe steps for characterizing hGBECs and their differentiation into hepatocytes. We then detail procedures to assess the functionality of the derived hepatocytes using key hepatic function assays.For complete details on the use and execution of this protocol, please refer to Chen et al.1

Cholangiocarcinoma (CCA) is a biologically diverse and highly aggressive cancer that arises from the biliary epithelium. It is typically divided into intrahepatic, perihilar, and distal types, each with distinct clinical behavior, genetic alterations, and therapeutic responses. Worldwide, the global incidence of CCA has risen steadily, accounting for nearly 15% of liver cancers and ∼3% of all gastrointestinal malignancies. CCA often presents at an advanced stage due to its silent onset and shows poor responsiveness to conventional chemotherapy, resulting in high mortality, accounting for ∼2% of cancer-related deaths worldwide. Risk factors include parasitic infections like liver flukes and chronic biliary diseases such as cholelithiasis and primary sclerosing cholangitis, although most cases have unknown origins. While early-stage patients may benefit from surgical resection or liver transplantation, these options are often not viable in advanced disease due to high relapse rates. In cases of unresectable or metastatic CCA, treatment remains difficult due to resistance and a lack of effective targeted therapies. This review systematically integrates the genomic, epigenetic, and signaling network mechanisms underlying CCA with their translational implications, providing a critical synthesis of the rapidly evolving field of targeted therapies, including recently approved Food and Drug Administration treatments and emerging novel agents. We specifically emphasize the key mechanisms of therapeutic resistance and corresponding strategies to overcome them, present an updated evaluation of vulnerabilities across distinct molecular subgroups, and explore the major challenges and future trajectories for advancing biomarker-driven precision medicine in CCA, thereby offering a forward-looking and clinically relevant perspective.

Understanding the liver stem cells (LSCs) holds great promise for new insights into liver diseases and liver regeneration. However, the heterogenicity and plasticity of liver cells have made it controversial. Here, by employing single-cell RNA-sequencing technology, transcriptome features of Krt19+ bile duct lineage cells isolated from Krt19CreERT; Rosa26R-GFP reporter mouse livers are examined. Distinct biliary epithelial cells which include adult LSCs, as well as their downstream hepatocytes and cholangiocytes are identified. Importantly, a novel cell surface LSCs marker, CD63, as well as CD56, which distinguished active and quiescent LSCs are discovered. Cell expansion and bi-potential differentiation in culture demonstrate the stemness ability of CD63+ cells in vitro. Transplantation and lineage tracing of CD63+ cells confirm their contribution to liver cell mass in vivo upon injury. Moreover, CD63+CD56+ cells are proved to be activated LSCs with vigorous proliferation ability. Further studies confirm that CD63+CD56- quiescent LSCs express VEGFR2 and FGFR1, and they can be activated to proliferation and differentiation through combination of growth factors: VEGF-A and bFGF. These findings define an authentic adult liver stem cells compartment, make a further understanding of fate regulation on LSCs, and highlight its contribution to liver during pathophysiologic processes.

The presence of neutrophil infiltration around the pancreatic ducts has been found to be associated with type 2 autoimmune pancreatitis (AIP). However, the functional role and clinical significance of neutrophil migration in the progression of pancreatitis is not fully understood. Here, we found that neutrophil extracellular traps (NETs) are abundant around the pancreatic duct in patients with type 2 AIP. We also observed an increased expression of toll-like receptor 9 (TLR9) in pancreatic ductal epithelial cells (HPDEC) in type 2 AIP patients compared to other pancreatic diseases. TLR9 acts as the DNA component of NETs (NET-DNA) receptor in HPDEC, which senses extracellular DNA and subsequently activates the NF-κB pathway to promote neutrophil recruitment and induce NET formation. In addition, our results indicated that the hydroxychloroquine (HCQ), acting as a TLR9 antagonist, could effectively inhibit the activation of inflammatory pathways, reduce neutrophil migration and block the positive feedback loop. The intervention positions HCQ acts as a potential target drug for the clinical treatment of type 2 AIP.

AbstractChronic pancreatitis (CP) is a complex disease with genetic and environmental factors at play. Through trio exome sequencing, a de novo SEC16A frameshift variant in a Chinese teenage CP patient is identified. Subsequent targeted next‐generation sequencing of the SEC16A gene in 1,061 Chinese CP patients and 1,196 controls reveals a higher allele frequency of rare nonsynonymous SEC16A variants in patients (4.90% vs 2.93%; odds ratio [OR], 1.71; 95% confidence interval [CI], 1.26–2.33). Similar enrichments are noted in a French cohort (OR, 2.74; 95% CI, 1.67–4.50) and in a biobank meta‐analysis (OR, 1.16; 95% CI, 1.04–1.31). Notably, Chinese CP patients with SEC16A variants exhibit a median onset age 5 years earlier than those without (40.0 vs 45.0; p = 0.012). Functional studies using three CRISPR/Cas9‐edited HEK293T cell lines show that loss‐of‐function SEC16A variants disrupt coat protein complex II (COPII) formation, impede secretory protein vesicles trafficking, and induce endoplasmic reticulum (ER) stress due to protein overload. Sec16a+/− mice, which demonstrate impaired zymogen secretion and exacerbated ER stress compared to Sec16a+/+, are further generated. In cerulein‐stimulated pancreatitis models, Sec16a+/− mice display heightened pancreatic inflammation and fibrosis compared to wild‐type mice. These findings implicate a novel pathogenic mechanism predisposing to CP.

Background & AimsApoptosis generates plenty of membrane-bound nanovesicles, the apoptotic vesicles (apoVs), which show promise for biomedical applications. The liver serves as a significant organ for apoptotic material removal. Whether and how the liver metabolizes apoptotic vesicular products and contributes to liver health and disease is unrecognized.MethodsapoVs were labeled and traced after intravenous infusion. Apoptosis-deficient mice by Fas mutant (Fasmut) and Caspase-3 knockout (Casp3−/−) were used with apoV replenishment to evaluate the physiological apoV function. Combinations of morphologic, biochemical, cellular, and molecular assays were applied to assess the liver while hepatocyte analysis was performed. Partial hepatectomy and acetaminophen liver failure models were established to investigate liver regeneration and disease recovery.ResultsWe discovered that the liver is a major metabolic organ of circulatory apoVs, in which apoVs undergo endocytosis by hepatocytes via a sugar recognition system. Moreover, apoVs play an indispensable role to counteract hepatocellular injury and liver impairment in apoptosis-deficient mice upon replenishment. Surprisingly, apoVs form a chimeric organelle complex with the hepatocyte Golgi apparatus through the soluble N-ethylmaleimide–sensitive factor attachment protein receptor machinery, which preserves Golgi integrity, promotes microtubule acetylation by regulating α-tubulin N-acetyltransferase 1, and consequently facilitates hepatocyte cytokinesis for liver recovery. The assembly of the apoV-Golgi complex is further revealed to contribute to liver homeostasis, regeneration, and protection against acute liver failure.ConclusionsThese findings establish a previously unrecognized functional and mechanistic framework that apoptosis through vesicular metabolism safeguards liver homeostasis and regeneration, which holds promise for hepatic disease therapeutics.

Abstract Background Liver ischemia/reperfusion (I/R) injury is usually caused by hepatic inflow occlusion during liver surgery, and is frequently observed during war wounds and trauma. Hepatocyte ferroptosis plays a critical role in liver I/R injury, however, it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase (DDX/DHX) family. Methods The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis. Hepatocyte-specific Dhx58 knockout mice were constructed, and a partial liver I/R operation was performed. Single-cell RNA sequencing (scRNA-seq) in the liver post I/R suggested enhanced ferroptosis by Dhx58hep−/−. The mRNAs and proteins associated with DExH-box helicase 58 (DHX58) were screened using RNA immunoprecipitation-sequencing (RIP-seq) and IP-mass spectrometry (IP-MS). Results Excessive production of reactive oxygen species (ROS) decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis, while treatment using IFN-α increased DHX58 expression and prevented ferroptosis during liver I/R injury. Mechanistically, DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4 (GPX4), a central ferroptosis suppressor, and recruits the m6A reader YT521-B homology domain containing 2 (YTHDC2) to promote the translation of Gpx4 mRNA in an m6A-dependent manner, thus enhancing GPX4 protein levels and preventing hepatic ferroptosis. Conclusions This study provides mechanistic evidence that IFN-α stimulates DHX58 to promote the translation of m6A-modified Gpx4 mRNA, suggesting the potential clinical application of IFN-α in the prevention of hepatic ferroptosis during liver I/R injury.

Interleukin-6 (IL-6) is a pleiotropic cytokine and exerts its complex biological functions mainly through three different signal modes, called cis-, trans-, and cluster signaling. When IL-6 binds to its membrane or soluble receptors, the co-receptor gp130 is activated to initiate downstream signaling and induce the expression of target genes. In the liver, IL-6 can perform its anti-inflammatory activities to promote hepatocyte reprogramming and liver regeneration. On the contrary, IL-6 also exerts the pro-inflammatory functions to induce liver aging, fibrosis, steatosis, and carcinogenesis. However, understanding the roles and underlying mechanisms of IL-6 in liver physiological and pathological processes is still an ongoing process. So far, therapeutic agents against IL‑6, IL‑6 receptor (IL‑6R), IL-6-sIL-6R complex, or IL-6 downstream signal transducers have been developed, and determined to be effective in the intervention of inflammatory diseases and cancers. In this review, we summarized and highlighted the understanding of the double-edged effects of IL-6 in liver homeostasis, aging, inflammation, and chronic diseases, for better shifting the "negative" functions of IL-6 to the "beneficial" actions, and further discussed the potential therapeutic effects of targeting IL-6 signaling in the clinics.

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终末期肝病包括肝衰竭和失代偿期肝硬化等类型,临床表现为肝细胞功能异常而导致肝脏蛋白合成、代谢等功能丧失.在我国,每年约有30万人死于终末期肝脏疾病.原位肝移植是治疗终末期肝病最为直接且有效的方式,但由于供肝资源短缺、免疫排斥和手术费昂贵等问题的存在,极大限制了其在临床上的广泛应用.细胞治疗是潜在的能够替代肝移植的唯一有希望的治疗方法,其主要是通过将体外获得的细胞移植入受体肝脏中,植入肝脏的外源细胞进行增殖并逐渐再殖受损肝脏从而实现损伤肝脏功能的修复.本文拟对有望成为细胞治疗的供体细胞如原代或诱导型肝细胞、肝脏干/前体细胞、间充质干细胞、肝脏类器官等细胞类型在动物实验和临床应用中取得的进展进行概述,为其应用于终末期肝脏疾病的临床治疗提供理论基础.

目的 探讨慢性损伤模型细胞周期抑制因子P21(P21)表达对肝损伤程度和胆管上皮细胞转分化为肝细胞的影响.方法 利用谱系示踪技术建立胆管上皮细胞示踪模型小鼠;在胆管上皮细胞示踪小鼠,利用腺相关病毒构建肝细胞特异性P21 过表达模型;利用胆碱缺乏/乙硫氨酸补充饮食(CDE)构建小鼠严重慢性肝损伤模型;采用蛋白印迹和免疫组织荧光法检测胆管上皮细胞标记和P21 过表达水平,以过表达P21 为实验组,过表达空载体为对照组,取损伤2w和损伤后恢复2w的小鼠肝组织,采用蛋白印迹、免疫组织化学和免疫组织荧光等技术检测肝损伤程度、细胞增殖水平、胆管上皮细胞转分化效果和转分化细胞特性.结果 在CDE诱导慢性肝损伤小鼠,肝细胞特异性过表达P21 组肝脏外观表面失去原有光泽,肝质量比为(4.5±0.1)%,显著低于对照组[(5.2±0.2)%,P<0.05];血清AST、ALT和TBIL水平分别为(385.4±12.7)U/L、(423.8±32.4)U/L和(21.3±1.4)μmol/L,显著高于对照组[分别为(175.9±11.4)U/L、(214.8±23.5)U/L和(10.5±0.9)μmol/L,P<0.05];过表达P21 组肝细胞Ki67 阳性比例为(0.1±0.1)%,显著低于对照组[(8.2±1.5)%,P<0.05],S期标志蛋白CyclinA2 阳性比例为(0.1±0.2)%,显著低于对照组[(3.2±0.7)%,P<0.05],而两组间G1 期特异性蛋白CyclinD1 阳性比例无显著性差异[(43.2±3.5)%对(42.0±2.8)%,P>0.05]],提示肝细胞过表达细胞周期抑制蛋白P21 使肝细胞阻滞于G1/S期;肝细胞过表达细胞周期抑制蛋白P21 组在损伤恢复 2 周时出现了带有绿色荧光标记的肝细胞样克隆,这群细胞表达HNF4α肝细胞标志物.进一步分析发现绿色肝细胞样克隆具有成熟肝细胞的特征,比如表达Alb、CYP3A4、CYP2E1 和CYP2D6 等.结论 在肝损伤过程中,P21 过表达抑制肝细胞增殖能力,导致肝损伤程度加剧,从而促进胆管上皮细胞转分化为具有功能的成熟肝细胞.

目的 通过小分子化合物改变细胞命运,诱导人胆囊上皮细胞(hGBEC)分化为具有功能的肝细胞样细胞.方法 在基质胶中对原代hGBEC进行三维培养,生长培养基中添加B27添加剂、N2添加剂、N-乙酰半胱氨酸、表皮生长因子、肝细胞生长因子等.添加小分子化合物/蛋白因子对细胞进行诱导分化,筛选出关键作用因子.采用PCR、qPCR和免疫荧光染色检测干细胞标志物及肝细胞相关标志物的表达情况,通过脂肪BODIPY-493染色、糖原过碘酸希夫染色和白蛋白ELISA检测评估细胞的肝样功能.结果 三维培养的hGBEC表达造血干细胞抗原CD133、上皮细胞黏附分子、肝细胞核因子4α等肝脏干细胞和肝前体细胞标志物.TGF-β信号通路抑制剂和Notch信号通路抑制剂是诱导hGBEC分化的关键作用因子.分化条件下所得细胞表达肝细胞功能标志物α1-抗胰蛋白酶、细胞色素P4503A4、白蛋白和延胡索酰乙酰乙酸水解酶,可以贮存糖原,具有合成脂肪的能力,能够分泌白蛋白.结论 hGBEC可在体外长期培养,通过抑制TGF-β和Notch信号通路可初步诱导其分化为具有部分肝功能的肝细胞样细胞.

细胞工程旨在通过相关理论让学生掌握如何科学改造细胞以获取目的产物,注重培养学生的科研思维、解决问题的能力和实践操作.在以往授课过程中,教师发现多数学生对课程相关领域的知识储备有限.文章介绍了案例教学在细胞工程中的具体实施方案与授课效果,通过围绕"诱导型多能干细胞的获取与应用"这一主题,安排课前资料查阅、课堂回答讨论、教师答疑与总结等模块完成授课,最后通过问卷调查、成绩评测分析教学效果.新的教学模式激起了学生的学习热情,锻炼了学生的探索能力,培养了专业思维素养,并延续了学生对所学专业的兴趣.今后,教师可通过凝练专业知识结合拓展具体应用,继续在该课程中开展案例教学.