Bisphenol AF (BPAF) is a widespread endocrine disruptor in the environment, and the use of BPAF has been strongly associated with the development of several diseases. In this study, we investigated the effects of BPAF on growth, development, oxidative stress and lipid metabolism in zebrafish. We chose the concentrations based on the measured LC50 at 96 h post-fertilization (96 hpf), and the zebrafish embryos were exposed to three different concentrations (0.125, 0.5 and 2 μmol/L). The findings indicated that BPAF exposure in zebrafish leaded to alterations in heart rate, body length and hatching rate, as well as an accumulation of red blood cells in the heart. Additionally, BPAF exposure resulted in increased levels of neutrophils, reactive oxygen species (ROS) and malondialdehyde (MDA), and decreased activity of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)), thus disturbing the balance between oxidative and antioxidative systems. BPAF promoted fatty acid catabolism and inhibited fatty acid synthesis, ultimately leading to a reduction in fatty acid content. Mechanistically, RNA-seq analysis and RT-qPCR revealed a significant upregulation of heat shock protein 70 (hsp70) after BPAF exposure. Inhibition of hsp70 with VER-155008 ameliorated BPAF-induced oxidative stress. These data provided a novel approach to investigate BPAF-induced oxidative stress and suggested that regulation of hsp70 is a crucial target for alleviating this process.

Cancer is a complicated disease that results from the interplay between specific changes in cellular genetics and diverse microenvironments. The application of high-performance and customizable clustered regularly interspaced palindromic repeats/associated protein (CRISPR/Cas) nuclease systems has significantly enhanced genome editing for accurate cancer modeling and facilitated simultaneous genetic modification for cancer therapy and mutation identification. Achieving an effective CRISPR/Cas platform for cancer treatment depends on the identification, selection, and optimization of specific mutated genes in targeted cancer tissues. However, overcoming the off-target effects, specificity, and immunogenicity are additional challenges that must be addressed while developing a gene editing system for cancer therapy. From this perspective, we briefly covered the pipeline of CRISPR/Cas cancer therapy, identified target genes to optimize gRNAs and sgRNAs, and explored alternative delivery modalities, including viral, non-viral, and extracellular vesicles. In addition, the list of patents and current clinical trials related to this unique cancer therapy method is discussed. In summary, we have discussed comprehensive start-to-end pipeline strategies for CRISPR/Cas development to advance the precision, effectiveness, and safety of clinical applications for cancer therapy.

Clinical drug screening is a critical component in disease treatment and in elucidating the impact of drug monitoring on cellular-level biochemical reactions. Field effect transistor (FET) based biosensors represent a novel technology in biomedical research, playing an essential role in clinical drug screening due to their high specificity, sensitivity, label-free detection, real-time monitoring, cost-effectiveness, and integration capabilities. This review provides a detailed description of the structural mechanisms of various types of FET biosensors. Furthermore, it introduces the classification, structural mechanisms, and immobilization methods of biological probes. Additionally, the methodologies and effects of utilizing the FET biosensor platform for screening cancer drugs, anti-infective drugs, inhibitor drugs, anti-histamine drugs, psychotropic drugs, cardiovascular drugs, and other drug categories are highlighted. Finally, the challenges encountered by FET biosensors in drug screening applications are discussed, and advanced strategies for future clinical drug screening are proposed.

CRISPR-based genetically modified scaffold-free biomaterials, including extracellular vehicles, cell sheets, cell aggregates, organoids and organs, have attracted significant attention in the fields of regenerative medicine and tissue engineering in recent years. With a wide range of applications in gene therapy, modeling disease, tissue regeneration, organ xenotransplantation, modeling organogenesis as well as gene and drug screening, they are at a critical juncture from clinical trials to therapeutic applications. Xenografts have already been tested on non-human primates and humans. However, we have to admit that a series of obstacles still need to be addressed, such as immune response, viral infection, off-target effects, difficulty in mass production, and ethical issues. Therefore, future research should pay more attention to improving their safety, accuracy of gene editing, flexibility of production, and ethical rationality. This review summarizes various types of CRISPR-based genetically modified scaffold-free biomaterials, including their preparation procedures, applications, and possible improvements.

Nonalcoholic fatty liver disease (NAFLD) is characterized by increased lipid accumulation and excessive deposition of extracellular matrix (ECM) that results in tissue stiffening. The potential interplay between matrix stiffness and hepatocyte lipid accumulation during NAFLD has not been established. Here, an in vitro NAFLD model is developed using chemically defined, engineered hydrogels and human induced pluripotent stem cell-derived hepatic organoids (HOs). Specifically, dynamic covalent chemistry crosslinking, along with transient small molecule competitors, are used to create dynamic stiffening hydrogels that enable the reproducible culture of HOs. Within matrices that mimic the stiffness of healthy to diseased tissue (≈1-6 kPa), lipid droplet accumulation in HOs is triggered by exposure to an NAFLD-associated free fatty acid. These NAFLD model suggests that higher stiffness microenvironments result in increased hepatic lipid droplet accumulation, increased expression of fibrosis markers, and increased metabolic dysregulation. By targeting the ROCK mechanosignaling pathway, the synergy between matrix stiffness and lipid droplet accumulation is disrupted. The in vitro model of NAFLD has the potential to understand the role of mechanosignaling in disease progression and identify new pathways for therapeutic intervention.

Excessive hepatic lipid accumulation is the hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD), yet its underlying mechanisms still not fully understood. In this study, we identified RNA binding motif protein 39 (Rbm39) as a key modulator of hepatic lipid homeostasis during MASLD progression. To establish in vivo MASLD model, mice were fed either a high-fat diet (HFD) or a Gubra-Amylin NASH (GAN) diet. We employed adeno-associated virus to manipulate Rbm39 expression levels to assess its role in MASLD. Transcriptome analysis was conducted to pinpoint the genes targeted by Rbm39. Western blot, RT-PCR, dual-luciferase reporter gene assays, and alternative splicing analysis were utilized to delve into the molecular mechanisms. Our results showed that Rbm39 expression was notably decreased in the livers of MASLD mice. Knockdown of hepatic Rbm39 aggravated HFD-induced hepatic steatosis and GAN diet-induced MASH, along with a notable decrease in serum lipid levels. Conversely, overexpression of Rbm39 attenuated MASLD development and progression. RNA sequencing data analysis indicated that Rbm39 regulated the expression of apolipoprotein B (Apob) and fatty acid-binding protein 4 (Fabp4), both of which are crucial for lipid transport. Mechanistically, Rbm39 enhanced the transcription of Apob by upregulating hepatocyte nuclear factor 4α (Hnf4α), while it suppressed Fabp4 transcription by regulating alternative splicing of hypoxia inducible factor-1α (Hif-1α). These findings highlight the pivotal role of Rbm39 in maintaining hepatic lipid homeostasis and suggest its potential as a therapeutic target for MASLD.

The liver plays a pivotal role in metabolism, detoxification, and protein synthesis and comprises several cell types, including hepatocytes and cholangiocytes. Primary human hepatocytes in 2D cultures rapidly dedifferentiate and lose their function, making their use as a reliable cell model challenging. Therefore, developing robust three-dimensional cell culture models is crucial, especially for diseases lacking reliable animal models. The aim of this study was to optimize a protocol for the directed differentiation of induced pluripotent stem cells into liver progenitor cells, achieving the high-level expression of specific markers. As a result, we established a 2D culture of liver progenitor cells capable of differentiating into three cell types: a 3D organoid culture containing hepatocyte- and cholangiocyte-like cells and a 2D cell culture comprising stellate-like cells. To evaluate gene delivery efficiency, liver progenitor cells were transduced with various rAAV serotypes carrying an eGFP reporter cassette at different multiplicities of infection (MOIs). Our results revealed that rAAV serotype 2/2 at MOI of 100,000 achieved the highest transduction efficiency of 93.6%, while electroporation demonstrated a plasmid delivery efficiency of 54.3%. These findings suggest that liver progenitor cells are a promising tissue-like cell model for regenerative medicine and demonstrate high amenability to genetic manipulation, underscoring their potential in gene therapy and genome editing studies.

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.

Metabolic dysfunction-associated steatotic liver disease (MASLD) remains the most common chronic liver disease worldwide, and appropriate in vitro models are of great significance for investigating pathogenesis and drug screening of MASLD. In this study, human expandable cholangiocyte organoids were derived from adult stem cells of normal liver tissue. After differentiation, liver organoids (LOs) exhibited the functional characteristics and genomic features of mature hepatocytes. To induce steatosis, LOs were incubated with a gradient concentration oleic acid, and it was found that the model could recapitulate the development of lipid accumulation and inflammation. In addition, the drug sensitivity of the hepatic steatosis model was further verified through anti-steatosis drug testing. In summary, LOs have great potential for disease modeling, and the results indicate that the hepatic steatosis model may serve as a useful tool for exploring the molecular mechanisms and drug screening of MASLD.

Selenoprotein I (SELENOI) was known initially as ethanolamine phosphotransferase 1 (EPT1) and later as a selenoprotein. Because global knockout of Selenoi in mice is embryonically lethal, we generated liver-specific Selenoi knockout (cKO) mice to reveal functions and mechanism of SELENOI in the liver. Compared with control mice, cKO mice (8 weeks old) had no differences in body weight, glucose metabolism, energy expenditure, overall health status, or liver histology. However, these mice had lower (P < 0.05) mRNA levels of 13 selenoprotein genes, contents of Se, GSH, and T-AOC (12-40%), and activities of antioxidant enzymes (17-51%), but higher (P < 0.05) mRNA levels of oxidative stress-related genes (34%-46%) in the liver than the control mice. They had a higher (P < 0.05) ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) due to increases of the former and decreases of the latter, altered PE and PC constituents such as n-6/n-3 PUFA ratios, and elevated mRNA levels (95%-2-fold, P < 0.05) of lipolysis genes, compared with the control mice. The knockout attenuated hepatic injury and fibrosis induced by 14 intraperitoneal injections of CCl4 (0.5 mL/kg). The protection was associated with adaptive cytoprotective mechanisms induced by the overall decline of redox status mediated by SELENOI as a selenoprotein and activations of PPAR signaling, fatty acid desaturase 2 (FADS2), glutathione S-transferase, and lipid peroxide hydrolysis through modulating biosynthesis and(or) constituents of PC, PE, and n-6/n-3 PUFAs mediated by SELENOI as EPT1. Inhibition of FADS2 in CCl4-treated cKO hepatocytes partially removed the protection by the knockout. In conclusion, hepatic SELENOI expression was not essential for survival, but served as a multifunctional regulator of hepatic selenogenome expression, Se metabolism, redox status, biosyntheses and profiles of PC and PE, and resistance to CCI4.

Ischemia-reperfusion injury stands as a primary instigator of acute kidney injury (AKI), prominently driven by oxidative stress. Among the critical antioxidant defenses is glutathione peroxidase 3 (GPX3), an enzyme generated by renal tubular epithelial cells. Our prior investigations have unveiled a substantial downregulation of GPX3 in renal tissues gleaned from AKI patients and murine models. This study aims to investigate the role of tubular cell-specific Gpx3 deletion on ischemia-reperfusion injury-induced AKI (IRI-AKI) in a murine model and delineate the potential underlying mechanisms. By generating renal tubular epithelial cell-specific Gpx3 knockout mice and inducing IRI-AKI, we assessed a spectrum of kidney injury indices including renal function, oxidative stress, apoptosis and mitochondrial dynamics. Additionally, we conducted transcriptome sequencing and bioinformatics analyses. The outcomes underscore that the deficiency of GPX3 in tubular cells exacerbates tubular injury, renal dysfunction, oxidative stress, apoptosis, and mitochondrial dynamic disturbances in the context of IRI-AKI. Sequencing and bioinformatics analysis suggest that the Gpx3 deletion predominantly impacts pathways associated with metabolism and inflammation. In conclusion, the tubular cell-specific deficiency of GPX3 exacerbates renal injury by intensifying oxidative stress, fostering mitochondrial impairment, perturbing metabolic processes and fueling inflammation. The targeted restoration of GPX3 in the renal tubular emerges as a potential therapeutic avenue for mitigating IRI-AKI.

Non-alcoholic fatty liver disease (NAFLD) is a global health concern with limited treatment options. The paucity of predictive i n v itro models in preclinical settings seems to be one of the limitations of identifying effective medicines. We therefore aimed to develop an i n v itro model that can display the key hallmarks of NAFLD, such as steatosis, inflammation, and fibrosis.

An in vitro model of steatohepatitis was developed using organoids prepared from hepatocytes of healthy individuals from a commercial source (HLOs) and the liver tissues collected from needle biopsies of NAFLD patients (HLONAFLD) using defined culture conditions. HLOs were treated with palmitic acid for 72 h to develop an i n v itro model of steatohepatitis, while HLONAFLD served as a natural model of steatohepatitis. Metformin and saroglitazar were used to validate the liver organoid model of steatohepatitis. Saroglitazar was also evaluated in the high-fat, high-fructose (HF-HF) diet-induced model of NAFLD using C57BL/6 mice to validate the findings from the i n v itro model.

HLOs and HLONAFLD exhibited bipotent properties, showing the expression of markers of hepatocytes, ductal cells, and also stem cells. Furthermore, they demonstrated the expression of nonparenchymal cell markers such as stellate cells (CD166) and Kupffer-like cells (CD68 and EMR1). The steatohepatitis models developed using these organoids displayed markers associated with steatosis, inflammation and fibrosis, which were decreased by metformin and saroglitazar.

The in vitro models developed in our lab employing HLOs and HLONAFLD display all three key hallmarks of NAFLD: steatosis, inflammation, and fibrosis without the necessity for coculture with other nonparenchymal cells. The implementation of the HLONAFLD-based model is also expected to provide a more realistic assessment of test substances to develop therapeutics for NAFLD.

The liver fulfils a plethora of vital functions and, due to their importance, liver dysfunction has life-threatening consequences. Liver disorders currently account for more than two million deaths annually worldwide and can be classified broadly into three groups, considering their onset and aetiology, as acute liver diseases, inherited metabolic disorders and chronic liver diseases. In the most advanced and severe forms leading to liver failure, liver transplantation is the only treatment available, which has many associated drawbacks, including a shortage of organ donors. Cell therapy via fully mature cell transplantation is an advantageous alternative that may be able to restore a damaged organ's functionality or serve as a bridge until regeneration can occur. Pioneering work has shown that transplanting adult hepatocytes can support liver recovery. However, primary hepatocytes cannot be grown extensively in vitro as they rapidly lose their metabolic activity. Therefore, different cell sources are currently being tested as alternatives to primary cells. Human pluripotent stem cell-derived cells, chemically induced liver progenitors, or 'liver' organoids, hold great promise for developing new cell therapies for acute and chronic liver diseases. This Review focuses on the advantages and drawbacks of distinct cell sources and the relative strategies to address different therapeutic needs in distinct liver diseases.

Over the last decade, a plethora of organoid models have been generated to recapitulate aspects of human development, disease, tissue homeostasis, and repair. Organoids representing multiple tissues have emerged and are typically categorized based on their origin. Tissue-derived organoids are established directly from tissue-resident stem/progenitor cells of either adult or fetal origin. Starting from pluripotent stem cells (PSCs), PSC-derived organoids instead recapitulate the developmental trajectory of a given organ. Gene editing technologies, particularly the CRISPR-Cas toolbox, have greatly facilitated gene manipulation experiments with considerable ease and scalability, revolutionizing organoid-based human biology research. Here, we review the recent adaptation of CRISPR-based screenings in organoids. We examine the strategies adopted to perform CRISPR screenings in organoids, discuss different screening scopes and readouts, and highlight organoid-specific challenges. We then discuss individual organoid-based genome screening studies that have uncovered novel genes involved in a variety of biological processes. We close by providing an outlook on how widespread adaptation of CRISPR screenings across the organoid field may be achieved, to ultimately leverage our understanding of human biology.

Over a third of the global population is affected by obesity, fatty liver disease (Metabolic Dysfunction-Associated Steatotic Liver Disease, MASLD), and its severe form, MASH (Metabolic Dysfunction-Associated Steatohepatitis), which can ultimately progress to hepatocellular carcinoma (HCC). Recent advancements include therapeutics such as glucagon-like peptide 1 (GLP-1) agonists and neural/vagal modulation strategies for these disorders. Among the many pathways regulating these conditions, emerging insights into transforming growth factor-β (TGF-β) signaling highlight potential future targets through its role in pathophysiological processes such as adipogenesis, inflammation, and fibrosis. Vagus nerve innervation in the gastrointestinal tract is involved in satiety regulation and energy homeostasis, and vagus nerve stimulation has been applied in weight loss and diabetes. This review explores clinical trials in obesity, novel therapeutic targets, and the role of TGF-β signaling and vagus nerve modulation in obesity-related liver diseases and HCC.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent liver disorder worldwide and can progress to steatohepatitis. Elevated de novo lipogenesis (DNL) is a key contributor to hepatic steatosis. Fatty acid (FA) desaturation produces several unsaturated lipid isomers that are structurally very similar but have diverse biological functions. However, due to their structural similarity, many conventional mass spectrometry approaches cannot detect such metabolic alterations. Thus, we introduce the Skylite (Skyline-based lipid isomer retention time evaluation) workflow using conventional liquid chromatography-mass spectrometry (LC-MS) to identify important isomer features. Retention times of isomeric phosphatidylcholines are compared with the well-characterized human plasma reference standard NIST 1950. Retention time trends correlate well with fixed-charge derivatized FA in liquid chromatography and ozone-induced dissociation mass spectrometry data. The interpretation is supported by double bond diagnostic fragments in LC-MS/MS experiments of epoxidized hydrolyzed fatty acids. We investigate hepatic lipid profiles, focusing on esterified fatty acids in two mouse models of metabolic dysfunction-associated steatohepatitis (MASH). Out of 37 phosphatidylcholine sum compositions, the workflow identifies 123 lipid features. Importantly, CCl4-induced and melanocortin-4 receptor knockout mice on a western diet (WD) have significantly higher levels of mead acid, branched-chain fatty acid, and n-7 PUFA incorporated into phosphatidylcholines. While the MASH mouse liver tissues contain notable amounts of n-7 PUFA, no n-10 PUFA were detected, potentially indicating a unique desaturation pattern. The screening for altered lipid isomer profiles bridges the gap between high-throughput analyses and specialized structure-resolved techniques.

Advances in nucleic acid delivery have positioned the liver as a key target for gene therapy, with adeno-associated virus vectors showing long-term effectiveness in treating hemophilia. Steatotic liver disease (SLD), the most common liver condition globally, primarily results from metabolic dysfunction-associated and alcohol-associated liver diseases. In some individuals, SLD progresses from simple steatosis to steatohepatitis, cirrhosis, and eventually hepatocellular carcinoma, driven by a complex interplay of genetic, metabolic, and environmental factors. Genetic variations in various lipid metabolism-related genes, such as patatin-like phospholipase domain-containing protein 3 (PNPLA3), 17β-hydroxysteroid dehydrogenase type 13 (HSD17B13), and mitochondrial amidoxime-reducing component 1 (MTARC1), impact the progression of SLD and offer promising therapeutic targets. This review largely focuses on genes identified through clinical association studies, as they are more likely to be effective and safe for therapeutic intervention. While preclinical research continues to deepen our understanding of genetic factors, early-stage clinical trials involving gene-based SLD therapies, including transient antisense and small-molecule approaches, are helping prioritize therapeutic targets. Meanwhile, hepatocyte gene editing technologies are advancing rapidly, offering alternatives to transient methods. As such, gene-based therapies show significant potential for preventing the progression of SLD and enhancing long-term liver health.

The high failure rates in clinical drug development based on animal models highlight the urgent need for more representative human models in biomedical research. In response to this demand, organoids and organ chips were integrated for greater physiological relevance and dynamic, controlled experimental conditions. This innovative platform-the organoids-on-a-chip technology-shows great promise in disease modeling, drug discovery, and personalized medicine, attracting interest from researchers, clinicians, regulatory authorities, and industry stakeholders. This review traces the evolution from organoids to organoids-on-a-chip, driven by the necessity for advanced biological models. We summarize the applications of organoids-on-a-chip in simulating physiological and pathological phenotypes and therapeutic evaluation of this technology. This section highlights how integrating technologies from organ chips, such as microfluidic systems, mechanical stimulation, and sensor integration, optimizes organoid cell types, spatial structure, and physiological functions, thereby expanding their biomedical applications. We conclude by addressing the current challenges in the development of organoids-on-a-chip and offering insights into the prospects. The advancement of organoids-on-a-chip is poised to enhance fidelity, standardization, and scalability. Furthermore, the integration of cutting-edge technologies and interdisciplinary collaborations will be crucial for the progression of organoids-on-a-chip technology.

Apolipoprotein B (ApoB) is essential for the assembly and secretion of triglyceride (TG)-rich VLDL particles, and its dysfunction is linked to metabolic disorders, including dyslipidemia and liver steatosis. However, less attention has been paid to whether and how other apolipoproteins play redundant or compensatory roles when the ApoB function is compromised. Here, we investigated the effects of microsomal triglyceride transfer protein (MTP), which mediates lipidation of nascent ApoB, on ApoE function. We observed a paradoxical increase in ApoE secretion resulting from increased expression in MTP inhibitor (MTPi)-treated human hepatoma cells. This phenotype was recapitulated in APOB-knockout cells and was associated with impaired ApoB secretion. While MTP-dependent transfer of neutral lipids is dispensable for ApoE secretion, TG biosynthesis, redundantly catalyzed by DGAT1 and DGAT2, is required for efficient ApoE secretion in hepatoma cells. ApoE colocalizes with lipid droplets near the Golgi apparatus and mediates TG export in an ApoB-independent fashion. We found that simultaneous inhibition of both ApoE and ApoB, but not inhibition of either alone, led to TG accumulation in hepatoma cells, indicating that both proteins function redundantly to control TG content. Validation studies in primary human hepatocytes (PHHs) demonstrated DGAT2-dependent secretion of ApoE. While MTPi treatment did not elevate ApoE secretion, it induced increased sialylation of ApoE in the supernatants of PHHs. These results show that enhanced ApoE secretion compensates for the impaired ApoB function to maintain the lipid homeostasis, providing an alternative route to modulate lipid turnover in hepatoma cells.

In high-producing dairy cows periparturient negative energy balance (NEB) triggers body fat mobilization, resulting in elevated blood levels of non-esterified fatty acids (NEFAs). Blood is dominated by the saturated fatty acids (SFA) palmitic (C16:0) and stearic acid (C18:0), which are associated with a negative effect on oocyte developmental competence. In contrast, the monounsaturated fatty acid (MUFA) oleic acid is harmless and is able to counteract the negative effect of saturated NEFAs on in vitro maturing oocytes. Since preantral follicles lack oleic acid-rich follicular fluid, we hypothesized that preantral follicles and oocytes may benefit from oleic acid-rich fat supplementation during NEB. Eight-month pregnant Holstein Friesian heifers were randomly divided in two groups to receive a standard, palmitic acid-rich (CTR, n = 5), or rumen-protected oleic acid-rich (UNSAT, n = 6), periparturient fat supplementation until 4 weeks post-calving. NEFA, β-Hydroxybutyric acid and haptoglobin profiles in blood were monitored, and cumulus-oocyte-complexes (COCs) were via transvaginal ovum pick-up (OPU) collected at 8, 12, and 16 weeks postpartum for in vitro maturation, fertilization (day 0), and culture until day 8. Oleic acid supplementation increased C18:1 and reduced C16:0 levels in blood in comparison to CTR, during the peripartum period. Interestingly, the UNSAT group exhibited a 1.6-times higher oocyte yield in comparison to the CTR, but no difference in oocyte developmental competence between the groups. These findings suggest that peripartum oleic acid supplementation supports follicles and oocytes during NEB. Potential long-term benefits of oleic acid on fertility in dairy cows, in a higher number of animals, warrant further investigation.

Metabolic dysfunction-associated steatotic liver disease (MASLD) represents the most prevalent chronic liver disorder globally. Due to its intricate pathogenesis and the current lack of efficacious pharmacological interventions, there is a pressing need to discover novel therapeutic targets and agents for MASLD treatment. Herein, it is found that histone deacetylase 11 (HDAC11), a subtype of HDAC family, is markedly overexpressed in both in vitro and in vivo models of MASLD. Furthermore, the knockdown of HDAC11 is observed to mitigate lipid accumulation in hepatic cells. A highly selective HDAC11 inhibitor, B6, which exhibits favorable pharmacokinetic property and liver distribution, is further designed and synthesized. Integrating RNA-seq data with in vivo and in vitro experiments, B6 is found to inhibit de novo lipogenesis (DNL) and promote fatty acid oxidation, thus mitigating hepatic lipid accumulation and pathological symptoms in MASLD mice. Further omics analysis and experiments reveal that B6 enhances the phosphorylation of AMPKα1 at Thr172 through the inhibition of HDAC11, consequently modulating DNL and fatty acid oxidation in the liver. In summary, this study identifies HDAC11 as a potential therapeutic target in MASLD and reports the discovery of a highly selective HDAC11 inhibitor with favorable drug-like properties for the treatment of MASLD.

The engineering construction of the liver has attracted enormous attention. Organoids, as emerging miniature three-dimensional cultivation units, hold significant potential in the biomimetic simulation of liver structure and function. Despite notable successes, organoids still face limitations such as high variability and low maturity. To overcome these challenges, engineering strategies have been established to maintain organoid stability and enhance their efficacy, laying the groundwork for the development of advanced liver organoids. The present review comprehensively summarizes the construction of engineered liver organoids and their prospective applications in biomedicine. Initially, we briefly present the latest research progress on matrix materials that maintain the three-dimensional morphology of organoids. Next, we discuss the manipulative role of engineering technologies in organoid assembly. Additionally, we outline the impact of gene-level regulation on organoid growth and development. Further, we introduce the applications of liver organoids in disease modeling, drug screening and regenerative medicine. Lastly, we overview the current obstacles and forward-looking perspectives on the future of engineered liver organoids. We anticipate that ongoing innovations in engineered liver organoids will lead to significant advancements in medical applications.

Hepatocellular carcinoma (HCC) represents a global health challenge, and ranks among one of the most prevalent and deadliest cancers worldwide. Therapeutic advances have expanded the treatment armamentarium for patients with advanced HCC, but obstacles remain. Precision oncology, which aims to match specific therapies to patients who have tumours with particular features, holds great promise. However, its implementation has been hindered by the existence of numerous 'HCC influencers' that contribute to the high inter-patient heterogeneity. HCC influencers include tumour-related characteristics, such as genetic alterations, immune infiltration, stromal composition and aetiology, and patient-specific factors, such as sex, age, germline variants and the microbiome. This Review delves into the intricate world of HCC, describing the most innovative model systems that can be harnessed to identify precision and/or personalized therapies. We provide examples of how different models have been used to nominate candidate biomarkers, their limitations and strategies to optimize such models. We also highlight the importance of reproducing distinct HCC influencers in a flexible and modular way, with the aim of dissecting their relative contribution to therapy response. Next-generation HCC models will pave the way for faster discovery of precision therapies for patients with advanced HCC.

Liver diseases represent a growing global health challenge, and the increasing prevalence of obesity and metabolic disorders is set to exacerbate this crisis. To meet evolving regulatory demands, patient-specific in vitro liver models are essential for understanding disease mechanisms and developing new therapeutic approaches. Organoid models, which faithfully recapitulate liver biology, can be established from both non-malignant and malignant liver tissues, offering insight into various liver conditions, from acute injuries to chronic diseases and cancer. Improved understanding of liver microenvironments, innovative biomaterials, and advanced imaging techniques now facilitate comprehensive and unbiased data analysis, paving the way for personalised medicine. In this review, we discuss state-of-the-art patient-derived liver organoid models, recent technological advancements, and strategies to enhance their clinical impact.

Human organoids are a widely used tool in cell biology to study homeostatic processes, disease, and development. The term organoids covers a plethora of model systems from different cellular origins that each have unique features and applications but bring their own challenges. This review discusses the basic principles underlying organoids generated from pluripotent stem cells (PSCs) as well as those derived from tissue stem cells (TSCs). We consider how well PSC- and TSC-organoids mimic the different intended organs in terms of cellular complexity, maturity, functionality, and the ongoing efforts to constitute predictive complex models of in vivo situations. We discuss the advantages and limitations associated with each system to answer different biological questions including in the field of cancer and developmental biology, and with respect to implementing emerging advanced technologies, such as (spatial) -omics analyses, CRISPR screens, and high-content imaging screens. We postulate how the two fields may move forward together, integrating advantages of one to the other.

Organoid culture has emerged as a forefront technology in the life sciences field. As "in vitro micro-organs", organoids can faithfully recapitulate the organogenesis process, and conserve the key structure, physiological function and pathological state of the original tissue or organ. Consequently, it is widely used in basic and clinical studies, becoming important preclinical models for studying diseases and developing therapies. Here, we introduced the definition and advantages of organoids and described the development and advances in hepatobiliary organoids research. We focus on applying hepatobiliary organoids in benign and malignant diseases of the liver and biliary tract, drug research, and regenerative medicine to provide valuable reference information for the application of hepatobiliary organoids. Despite advances in research and treatment, hepatobiliary diseases including carcinoma, viral hepatitis, fatty liver and bile duct defects have still been conundrums of the hepatobiliary field. It is necessary and crucial to study disease mechanisms, establish efficient and accurate research models and find effective treatment strategies. The organoid culture technology shed new light on solving these issues. However, the technology is not yet mature, and many hurdles still exist that need to be overcome. The combination with new technologies such as CRISPR-HOT, organ-on-a-chip may inject new vitality into future development.

To date, generating viable and functional hepatocytes in large scale remains challenge. By employing 3D suspension condition with the support of low concentration Matrigel, a novel culture system was developed to generate expandable hepatoblast organoids (HB-orgs) and mature polarised hepatocyte organoids (P-hep-orgs) from human embryonic stem cells (hESCs) in both dishes and bioreactors. scRNA-seq and functional assays were used to characterise HB-orgs and P-hep-orgs. hESC-derived HB-orgs could proliferate at least for 15 passages, leading to 1012 in total cells in 4 weeks. P-hep-orgs differentiated from HB-orgs displayed characteristics of mature hepatocytes with polarisation. Moreover, single-cell RNA sequencing exhibited that over 40% of cells in P-hep-orgs were highly fidelity with human primary hepatocytes. Eventually, large-scale production of P-hep-orgs could be generated from massively expanded HB-orgs within 1 week with similar number in bioreactors, which were achieved by the enhancements in energy metabolism contribute to the expansion of HB-orgs and maturation of P-hep-orgs in bioreactors. By providing a cost-efficient and robust platform, our study represents a significant step toward manufacturing large-scale functioning hESC-derived hepatocytes for cell-based therapeutics, disease modelling, pharmacology and toxicology studies.

Developing disease models and screening for effective drugs are key areas of modern medical research. Traditional methodologies frequently fall short in precisely replicating the intricate architecture of bodily tissues and organs. Nevertheless, recent advancements in biomaterial-assisted organoid technology have ushered in a paradigm shift in biomedical research. This innovative approach enables the cultivation of three-dimensional cellular structures in vitro that closely emulate the structural and functional attributes of organs, offering physiologically superior models compared to conventional techniques. The evolution of biomaterials plays a pivotal role in supporting the culture and development of organ tissues, thereby facilitating more accurate disease state modeling and the rigorous evaluation of drug efficacy and safety profiles. In this review, we will explore the roles that various biomaterials play in organoid development, examine the fundamental principles and advantages of utilizing these technologies in constructing disease models, and highlight recent advances and practical applications in drug screening using disease-specific organoid models. Additionally, the challenges and future directions of organoid technology are discussed. Through continued research and innovation, we aim to make remarkable strides in disease treatment and drug development, ultimately enhancing patient quality of life.

Organoids are innovative three-dimensional and self-organizing cell cultures of various lineages that can be used to study diverse tissues and organs. Human organoids have dramatically increased our understanding of developmental and disease biology. They provide a patient-specific model to study known diseases, with advantages over animal models, and can also provide insights into emerging and future health threats related to climate change, zoonotic infections, environmental pollutants or even microgravity during space exploration. Furthermore, organoids show potential for regenerative cell therapies and organ transplantation. Still, several challenges for broad clinical application remain, including inefficiencies in initiation and expansion, increasing model complexity and difficulties with upscaling clinical-grade cultures and developing more organ-specific human tissue microenvironments. To achieve the full potential of organoid technology, interdisciplinary efforts are needed, integrating advances from biology, bioengineering, computational science, ethics and clinical research. In this Review, we showcase pivotal achievements in epithelial organoid research and technologies and provide an outlook for the future of organoids in advancing human health and medicine.

The creation of self-organizing liver organoids represents a significant, although modest, step toward addressing the ongoing organ shortage crisis in allogeneic liver transplantation. However, researchers have recognized that achieving a fully functional whole liver remains a distant goal, and the original ambition of organoid-based liver generation has been temporarily put on hold. Instead, liver organoids have revolutionized the field of hepatology, extending their influence into various domains of precision and molecular medicine. These 3D cultures, capable of replicating key features of human liver function and pathology, have opened new avenues for human-relevant disease modeling, CRISPR gene editing, and high-throughput drug screening that animal models cannot accomplish. Moreover, advancements in creating more complex systems have led to the development of multicellular assembloids, dynamic organoid-on-chip systems, and 3D bioprinting technologies. These innovations enable detailed modeling of liver microenvironments and complex tissue interactions. Progress in regenerative medicine and transplantation applications continues to evolve and strives to overcome the obstacles of biocompatibility and tumorigenecity. In this review, we examine the current state of liver organoid research by offering insights into where the field currently stands, and the pivotal developments that are shaping its future.

This study explored the evolving landscape of Microphysiological Systems (MPS), with a focus on organoids and organ-on-a-chip (OoC) technologies, which are promising alternatives to animal testing in drug discovery. MPS technology offers in vitro models with high physiological relevance, simulating organ function for pharmacokinetic studies. Organoids composed of 3D cell aggregates and OoCs mimicking in vivo environments based on microfluidic platforms represent the forefront of MPS. This paper provides a comprehensive overview of their application in studying the gut, liver, and kidney and their challenges in becoming reliable alternatives to in vivo models. Although MPS technology is not yet fully comparable to in vivo systems, its continued development, aided by in silico, automation, and AI approaches, is anticipated to bring about further advancements. Collaboration across multiple disciplines and ongoing regulatory discussions will be crucial in driving MPS toward practical and ethical applications in biomedical research and drug development.

The lack of in vivo accurate human liver models hinders the investigation of liver-related diseases, injuries, and drug-related toxicity, posing challenges for both basic research and clinical applications. Traditional cellular and animal models, while widely used, have significant limitations in replicating the liver's complex responses to various stressors. Liver organoids derived from human pluripotent stem cells, adult stem cells primary cells, or tissues can mimic diverse liver cell types, major physiological functions, and architectural features. Recent advancements in the field have shown that some liver organoids have sufficient accuracy to replicate specific aspects of the human liver's complexity. This review highlights recent progress in liver organoid research, with a particular emphasis on their potential for toxicity assessment and disease modeling. The intrinsic advantages of liver organoids include higher sensitivity and suitability for long-term studies, which enhance the predictive value in drug and nanomaterial toxicity testing. The integration of liver organoids with microfluidic devices enables the simulation of the liver microenvironment and facilitates high-throughput drug screening. The liver organoids also serve as ideal platforms for studying liver diseases such as hepatitis, liver fibrosis, viral liver diseases, and monogenic diseases. Additionally, this review discusses the advantages and limitations of liver organoids along with potential avenues for future research.

Chronic hepatobiliary damage progressively leads to fibrosis, which may evolve into cirrhosis and/or hepatocellular carcinoma. The fight against the increasing incidence of liver-related morbidity and mortality is challenged by a lack of clinically validated early-stage biomarkers and the limited availability of effective anti-fibrotic therapies. Current research is focused on uncovering the pathogenetic mechanisms that drive liver fibrosis. Drugs targeting molecular pathways involved in chronic hepatobiliary diseases, such as inflammation, hepatic stellate cell activation and proliferation, and extracellular matrix production, are being developed. Etiology-specific treatments, such as those for hepatitis B and C viruses, are already in clinical use, and efforts to develop new, targeted therapies for other chronic hepatobiliary diseases are ongoing. In this review, we highlight the major molecular changes occurring in patients affected by metabolic dysfunction-associated steatotic liver disease, viral hepatitis (Delta virus), and autoimmune chronic liver diseases (autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis). Further, we describe how this knowledge is linked to current molecular therapies as well as ongoing preclinical and clinical research on novel targeting strategies, including nucleic acid-, mesenchymal stromal/stem cell-, and extracellular vesicle-based options. Much clinical development is obviously still missing, but the plethora of promising potential treatment strategies in chronic hepatobiliary diseases holds promise for a future reversal of the current increase in morbidity and mortality in this group of patients.

Obesity is becoming one of the major non-communicable diseases with increasing incidence and risks that cannot be ignored. However effective and safe clinical treatment strategies still need to be deeply explored. Increased number and volume of adipocytes lead to overweight and obesity. The aim of our work is to identify lncRNAs that have important regulatory in differentiation of human mesenchymal stem cells (MSCs) into adipocytes, and to provide effective targets for clinical prevention and treatment of obesity and related metabolic disorders.

We extracted primary MSCs from human adipose tissue, and conducted expression profile analysis of lncRNAs during adipogenic differentiation of MSCs to screen changed lncRNAs. Characteristics of lncRNA were revealed mainly by RACE and RNA FISH. Loss- and gain-of function experiments in vivo and in vitro were used to analyze effects of lncRNA. Targeted metabolomics was utilized to detect levels of free fatty acids. RNA pull-down, mRNA stability tests, etc. were employed to explore mechanisms of lncRNA.

Human-specific lncRNA, we named it MEK6-AS1, was the most up-regulated transcript during adipogenic differentiation of MSCs. MEK6-AS1 was highly expressed in adipose tissue samples from individuals with BMI ≥ 25 and positively correlated with adipogenic marker genes in these samples. Knocking down lncRNA inhibited expression of adipogenic differentiation markers and ectopic adipogenesis, reducing contents of various free fatty acids, as well as promoting osteogenic differentiation. Overexpression of lncRNA had the opposite effects to the above processes. We also found that MEK6-AS1 was elevated during hepatic steatosis organoid generation. Mechanistically, MEK6-AS1 worked partially through stabilization of MEK6 mRNA by NAT10.

We have identified a human-specific lncRNA (MEK6-AS1) with position information in the genomic database but has not been extensively reported. We demonstrated that MEK6-AS1 as a novel lncRNA involved in adipogenic differentiation and adipogenesis, fatty acid metabolism, and osteogenic differentiation. We found that MEK6-AS1 may exert its effect by enhancing MEK6 mRNA stability through NAT10. Our study may provide insights into implication of lncRNAs in stem cell biology and offer a new potential therapeutic target for the prevention and treatment of obesity and other related disease.

Patient-derived models (PDMs, particularly organoids and xenografts) are irreplaceable tools for precision medicine, from target development to lead identification, then to preclinical evaluation, and finally to clinical decision-making. So far, PDM-based proteomics has emerged to be one of the cutting-edge directions and massive data have been accumulated. However, such PDM-based proteomic data have not been provided by any of the available databases, and proteomics profiles of all proteins in proteomic study are also completely absent from existing databases. Herein, an integrated database named 'OrgXenomics' was thus developed to provide the proteomic data for PDMs, which was unique in (a) explicitly describing the establishment detail for a wide array of models, (b) systematically providing the proteomic profiles (expression/function/interaction) for all proteins in studied proteomic analysis and (c) comprehensively giving the raw data for diverse organoid/xenograft-based proteomic studies of various diseases. Our OrgXenomics was expected to server as one good complement to existing proteomic databases, and had great implication for the practice of precision medicine, which could be accessed at: https://idrblab.org/orgxenomics/.

Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease with few therapeutic options. To narrow the translational gap in the development of pharmacological MASH treatments, a 3D liver model from primary human hepatocytes and non-parenchymal cells derived from patients with histologically confirmed MASH was established. The model closely mirrors disease-relevant endpoints, such as steatosis, inflammation and fibrosis, and multi-omics analyses show excellent alignment with biopsy data from 306 MASH patients and 77 controls. By combining high-content imaging with scalable biochemical assays and chemogenomic screening, multiple novel targets with anti-steatotic, anti-inflammatory, and anti-fibrotic effects are identified. Among these, activation of the muscarinic M1 receptor (CHRM1) and inhibition of the TRPM8 cation channel result in strong anti-fibrotic effects, which are confirmed using orthogonal genetic assays. Strikingly, using biosensors based on bioluminescence resonance energy transfer, a functional interaction along a novel MASH signaling axis in which CHRM1 inhibits TRPM8 via Gq/11 and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate can be demonstrated. Combined, this study presents the first patient-derived 3D MASH model, identifies a novel signaling module with anti-fibrotic effects, and highlights the potential of organotypic culture systems for phenotype-based chemogenomic drug target identification at scale.

In recent years, the number of people suffering from lifestyle diseases such as hyperlipidemia and fatty liver disease has increased rapidly due to westernization of dietary patterns. Among fatty liver diseases, those that are not caused by alcohol are referred to as nonalcoholic fatty liver disease (NAFLD). Some NAFLD can progress to nonalcoholic steatohepatitis (NASH), and further progression of NAFLD can lead to cirrhosis and liver cancer. Although numerous studies have demonstrated the efficacy of dietary polyunsaturated fatty acids (PUFAs), particularly omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), against NAFLD, the detailed mechanisms by which these PUFAs exert their protective effects on the pathogenesis and progression of NAFLD are not well understood. Recent studies using knockout mouse models and genome-wide association studies have suggested a potential role for the enzymes responsible for the biosynthesis of PUFAs (FADS1, FADS2, ELOVL2, and ELOVL5) and their incorporation into phospholipids (LPCAT3/MBOAT5/LPLAT12 and LPIAT1/MBOAT7/LPLAT11) in the development of NAFLD. In this review, we summarize recent findings on the association of NAFLD and PUFAs with a focus on PUFA biosynthetic and metabolic enzymes to discuss the potential role of PUFAs in the prevention of NAFLD.