Hepatic stellate cells (HSCs) have a central pathogenetic role in the development of liver fibrosis. However, their fibrosis-independent and homeostatic functions remain poorly understood1-5. Here we demonstrate that genetic depletion of HSCs changes WNT activity and zonation of hepatocytes, leading to marked alterations in liver regeneration, cytochrome P450 metabolism and injury. We identify R-spondin 3 (RSPO3), an HSC-enriched modulator of WNT signalling, as responsible for these hepatocyte-regulatory effects of HSCs. HSC-selective deletion of Rspo3 phenocopies the effects of HSC depletion on hepatocyte gene expression, zonation, liver size, regeneration and cytochrome P450-mediated detoxification, and exacerbates alcohol-associated and metabolic dysfunction-associated steatotic liver disease. RSPO3 expression decreases with HSC activation and is inversely associated with outcomes in patients with alcohol-associated and metabolic dysfunction-associated steatotic liver disease. These protective and hepatocyte-regulating functions of HSCs via RSPO3 resemble the R-spondin-expressing stromal niche in other organs and should be integrated into current therapeutic concepts.

Mesenchymal stromal cells (MSC) play a critical role in the stem cell niche, a specialized microenvironment where stem cells reside and interact with surrounding cells and extracellular matrix components. Within the niche, MSC offer structural support, modulate inflammatory response, promote angiogenesis and release specific signaling molecules that influence stem cell behavior, including self-renewal, proliferation and differentiation. In epithelial tissues such as the intestine, stomach and liver, MSC act as an important source of cytokines and growth factors, but not much is known about their role in the pancreas. Our group has established a standardized technology for the generation of pancreatic organoids. Herein, we investigated the role of pancreatic mesenchymal stromal cells in the regulation of human pancreatic organoid proliferation and growth, using this 3D model in a co-culture system. We particularly focused on the capacity of pancreatic MSC to produce R-spondin factors, which are considered critical regulators of epithelial growth. We propose the development of a complex in vitro system that combines organoid technology and mesenchymal stromal cells, thereby promoting the assembloid new research era.

Endoplasmic reticulum (ER) stores Ca2+ and plays crucial roles in protein folding, lipid transfer, and it's perturbations trigger an ER stress. In the liver, chronic ER stress is involved in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). Dysfunction of sarco/endoplasmic reticulum calcium ATPase (SERCA2), a key regulator of Ca2+ transport from the cytosol to ER, is associated with the induction of ER stress and lipid droplet formation. We previously identified NACHT and WD repeat domain-containing protein 1 (Nwd1) localized at the ER and mitochondria. However, the physiological significance of Nwd1 outside the brain remains unclear. In this study, we revealed that Nwd1-/- mice exhibited pathological manifestations comparable to MASH. Nwd1 interacts with SERCA2 near ER membranes. Nwd1-/- livers exhibited reduced SERCA2 ATPase activity and a smaller Ca2+ pool in the ER, leading to an exacerbated state of ER stress. These findings highlight the importance of SERCA2 activity mediated by Nwd1 in the pathogenesis of MASH.

Liver repair and regeneration are crucial physiological responses to hepatic injury and are orchestrated through intricate cellular and molecular networks. This review systematically delineates advancements in the field, emphasizing the essential roles played by diverse liver cell types. Their coordinated actions, supported by complex crosstalk within the liver microenvironment, are pivotal to enhancing regenerative outcomes. Recent molecular investigations have elucidated key signaling pathways involved in liver injury and regeneration. Viewed through the lens of metabolic reprogramming, these pathways highlight how shifts in glucose, lipid, and amino acid metabolism support the cellular functions essential for liver repair and regeneration. An analysis of regenerative variability across pathological states reveals how disease conditions influence these dynamics, guiding the development of novel therapeutic strategies and advanced techniques to enhance liver repair and regeneration. Bridging laboratory findings with practical applications, recent clinical trials highlight the potential of optimizing liver regeneration strategies. These trials offer valuable insights into the effectiveness of novel therapies and underscore significant progress in translational research. In conclusion, this review intricately links molecular insights to therapeutic frontiers, systematically charting the trajectory from fundamental physiological mechanisms to innovative clinical applications in liver repair and regeneration.

Organoids are miniature three-dimensional (3D) organ-like structures developed from primary cells that closely mimic the key histological, functional, and molecular characteristics of their parent organs. These structures self-organize through cell-cell and cell-matrix interaction in culture. In the last decade, organoids and allied 3D culture technologies have catalyzed studies involving developmental biology, disease biology, high-throughput drug screening, personalized medicine, biomarker discovery, tissue engineering, and regenerative medicine. Many organoid systems have been generated from the gastrointestinal system, for example, intestine, stomach, liver, pancreas, or colon. Gallbladder cancer (GBC) is the most common and highly aggressive form of biliary tract cancer. GBC is rare in the west but has a high incidence in South America and India. Prolonged chronic inflammation is implicated in the pathogenesis of GBC but the driving molecular pathways leading to neoplasia are not well understood. Gallbladder cholangiocyte organoids (GCO) will facilitate the understanding of the evolution of the disease and novel therapeutic strategies. In this review, we have discussed alternative methodologies and culture conditions developed to generate GCO models, applications that these models have been subjected to and the current limitations for the use of GCOs in addressing the challenges in GBC research.

Recently, we identified myeloid-derived growth factor (MYDGF) as a blood flow-induced angiocrine signal that promotes human and mouse hepatocyte proliferation and survival. Here, we review literature reporting changes in blood flow after partial organ resection in the liver, lung, and kidney, and we describe the angiocrine signals released by endothelial cells (ECs) upon blood flow alterations in these organs. While hepatocyte growth factor (HGF) and MYDGF are important angiocrine signals for liver regeneration, by now, angiocrine signals have also been reported to stimulate hyperplasia and/or hypertrophy during the regeneration of lungs and kidneys. In addition, angiocrine signals play a critical role in tumor growth. Understanding the mechano-elastic properties and flow-mediated alterations in the organ-specific microvasculature is crucial for therapeutic approaches to maintain organ health and initiate organ renewal.

Hepatocytes are organized into distinct zonal subsets across the liver lobule, yet their contributions to liver homeostasis and regeneration remain controversial. Here, we developed multiple genetic lineage-tracing mouse models to systematically address this. We found that the liver lobule can be divided into two major zonal and molecular hepatocyte populations marked by Cyp2e1 or Gls2. Pericentral Cyp2e1+ and periportal Gls2+ hepatocytes maintain their own lineage during adult homeostasis, while Cyp2e1+ hepatocytes fuel neonatal liver growth. The Gls2+ and Cyp2e1+ populations can rapidly regenerate one another when one of the populations is severely damaged. Midlobular Ccnd1+ hepatocytes are enriched in the Cyp2e1+ zone in adult liver but have limited contributions to regeneration upon partial hepatectomy and severe pericentral injury. Remarkably, Lgr5+ hepatocytes, a unique Cyp2e1+ subset, contribute significantly to liver replenishment upon periportal injuries. Our findings unravel that zonal hepatocytes mainly self-maintain during homeostasis but exhibit complex plasticity in repair upon injury.

During development, endothelial cells (ECs) undergo an extraordinary specialization by which generic capillary microcirculatory networks spanning from arteries to veins transform into patterned organotypic zonated blood vessels. These capillary ECs become specialized to support the cellular and metabolic demands of each specific organ, including supplying tissue-specific angiocrine factors that orchestrate organ development, maintenance of organ-specific functions and regeneration of injured adult organs. Here, we illustrate the mechanisms by which microenvironmental signals emanating from non-vascular niche cells induce generic ECs to acquire specific inter-organ and intra-organ functional attributes. We describe how perivascular, parenchymal and immune cells dictate vascular heterogeneity and capillary zonation, and how this system is maintained through tissue-specific signalling activated by vasculogenic and angiogenic factors and deposition of matrix components. We also discuss how perturbation of organotypic vascular niche cues lead to erasure of EC signatures, contributing to the pathogenesis of disease processes. We also describe approaches that use reconstitution of tissue-specific signatures of ECs to promote regeneration of damaged organs.

The unique architecture of the liver consists of hepatic lobules, dividing the hepatic features of metabolism into 2 distinct zones, namely the pericentral and periportal zones, the spatial characteristics of which are broadly defined as metabolic zonation. R-spondin3 (Rspo3), a bioactive protein promoting the Wnt signaling pathway, regulates metabolic features especially around hepatic central veins. However, the functional impact of hepatic metabolic zonation, regulated by the Rspo3/Wnt signaling pathway, on whole-body metabolism homeostasis remains poorly understood. In this study, we analyze the local functions of Rspo3 in the liver and the remote actions of hepatic Rspo3 on other organs of the body by using murine models. Rspo3 expression analysis shows that Rspo3 expression patterns are spatiotemporally controlled in the murine liver such that it locates in the pericentral zones and converges after feeding, and the dynamics of these processes are disturbed in obesity. We find that viral-mediated induction of Rspo3 in hepatic tissue of obesity improves insulin resistance and prevents body weight gain by restoring attenuated organ insulin sensitivities, reducing adipose tissue enlargement and reversing overstimulated adaptive thermogenesis. Denervation of the hepatic vagus suppresses these remote effects, derived from hepatic Rspo3 induction, toward adipose tissues and skeletal muscle, suggesting that signals are transduced via the neuronal communication consisting of afferent vagal and efferent sympathetic nerves. Furthermore, the non-neuronal inter-organ communication up-regulating muscle lipid utilization is partially responsible for the ameliorations of both fatty liver development and reduced skeletal muscle quality in obesity. In contrast, hepatic Rspo3 suppression through Cre-LoxP-mediated recombination system exacerbates diabetes due to glucose intolerance and insulin resistance, promotes fatty liver development and decreases skeletal muscle quality, resulting in obesity. Taken together, our study results reveal that modulation of hepatic Rspo3 contributes to maintaining systemic glucose metabolism and body composition via a newly identified inter-organ communication mechanism.

Rspondin 1 (Rspo1), a protein family member featuring secreted furin-like domains, plays a pivotal role in cancer development and exhibits a positive correlation with tumor progression. However, its expression in esophageal squamous cell carcinoma (ESCC) is still unknown.

Here, we assessed the correlation between Rspo1 and clinicopathological features of ESCC patients, and further investigated the potential role of Rspo1 in ESCC development and clinical outcomes.

This was a pilot study.

A total of 112 paraffin-embedded tumor samples from patients with ESCC, including 68 matched adjacent normal tissues, were collected post-surgery. Subsequently, tissue microarray (TMA) and immunohistochemistry (IHC) techniques were employed to assess the protein levels of Rspo1.

All statistical analyses were performed with SPSS 20.0 (SPSS, Inc., Chicago, IL).

We found that Rspo1 expression was significantly higher in ESCC than in adjacent normal tissues ( P < 0.0001). Moreover, Rspo1 was highly expressed in ESCC tumor specimens and showed a significant correlation with the T classification of ESCC ( P < 0.05). Additionally, our findings indicate a positive relationship between Rspo1 and survival time in ESCC. Patients exhibiting moderate to high levels of Rspo1 expression demonstrated superior survival outcomes compared to those with low expression ( P = 0.0002).

Our investigation has demonstrated that Rspo1 is upregulated in ESCC and exhibits a positive correlation with disease progression. Furthermore, we have observed a significant association between Rspo1 overexpression and improved patient survival rates, indicating its potential as a prognostic marker and therapeutic target for ESCC treatment.

ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the protein most negatively correlated with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptors, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.

Biliary obstruction and cholangiocyte hyperproliferation are important features of cholangiopathies affecting the large extrahepatic bile duct (EHBD). The mechanisms underlying obstruction-induced cholangiocyte proliferation in the EHBD remain poorly understood. Developmental pathways, including WNT signaling, are implicated in regulating injury responses in many tissues, including the liver. To investigate the contribution of WNT signaling to obstruction-induced cholangiocyte proliferation in the EHBD, we used complementary in vivo and in vitro models with pharmacologic interventions and transcriptomic analyses. To model obstruction, we used bile duct ligation (BDL) in mice. Human and mouse biliary organoids and mouse biliary explants were used to investigate the effects of WNT activation and inhibition in vitro. We observed an upregulation of WNT ligand expression associated with increased biliary proliferation following obstruction. Cholangiocytes were identified as both WNT ligand-expressing and WNT-responsive cells. Inhibition of WNT signaling decreased cholangiocyte proliferation in vivo and in vitro, while activation increased proliferation. WNT effects on cholangiocyte proliferation were β-catenin dependent, and we showed a direct effect of WNT7B on cholangiocyte growth. Our studies suggested that cholangiocyte-derived WNT ligands can activate WNT signaling to induce proliferation after obstructive injury. These findings implicate the WNT pathway in injury-induced cholangiocyte proliferation within the EHBD.

Hepatic ischemia-reperfusion (IR) injury is the most common complication that occurs in liver surgery and hemorrhagic shock. ATP citrate lyase (Acly) plays a pivotal role in chromatin modification via generating acetyl-CoA for histone acetylation to influence biological processes. We aim to examine the roles of Acly, which is highly expressed in hepatocytes, in liver IR injury.

The functions of Acly in hepatic IR injury were examined in the mouse model with a hepatocyte-specific knockout of Acly . The Acly target genes were analyzed by CUT&RUN assay and RNA sequencing. The relationship between the susceptibility of the steatotic liver to IR and Acly was determined by the gain of function studies in mice. Hepatic deficiency of Acly exacerbated liver IR injury. IR induced Acly nuclear translocation in hepatocytes, which spatially fueled nuclear acetyl-CoA. This alteration was associated with enhanced acetylation of H3K9 and subsequent activation of the Foxa2 signaling pathway. Nuclear localization of Acly enabled Foxa2-mediated protective effects after hypoxia-reperfusion in cultured hepatocytes, while cytosolic Acly demonstrated no effect. The presence of steatosis disrupted Acly nuclear translocation. In the steatotic liver, restoration of Acly nuclear localization through overexpression of Rspondin-1 or Rspondin-3 ameliorated the IR-induced injury.

Our results indicate that Acly regulates histone modification by means of nuclear AcCoA production in hepatic IR. Disruption of Acly nuclear translocation increases the vulnerability of the steatotic liver to IR. Nuclear Acly thus may serve as a potential therapeutic target for future interventions in hepatic IR injury, particularly in the context of steatosis.

The liver is the largest solid organ in the body and is primarily composed of hepatocytes (HCs), endothelial cells (ECs), Kupffer cells (KCs), and hepatic stellate cells (HSCs), which spatially interact and cooperate with each other to maintain liver homeostasis. However, the complexity and molecular mechanisms underlying the crosstalk between these different cell types remain to be revealed. Here, we generated mice with conditional deletion of Gdf2 (also known as Bmp9) and Bmp10 in different liver cell types and demonstrated that HSCs were the major source of GDF2 and BMP10 in the liver. Using transgenic ALK1 (receptor for GDF2 and BMP10) reporter mice, we found that ALK1 is expressed on KCs and ECs other than HCs and HSCs, and GDF2 and BMP10 secreted by HSCs promote the differentiation of KCs and ECs and maintain their identity. Pdgfb expression was significantly upregulated in KCs and ECs after Gdf2 and Bmp10 deletion, ultimately leading to HSCs activation and liver fibrosis. ECs express several angiocrine factors, such as BMP2, BMP6, Wnt2, and Rspo3, to regulate HC iron metabolism and metabolic zonation. We found that these angiocrine factors were significantly decreased in ECs from Gdf2/Bmp10HSC-KO mice, which further resulted in liver iron overload and disruption of HC zonation. In summary, we demonstrated that HSCs play a central role in mediating liver cell-cell crosstalk via the production of GDF2 and BMP10, highlighting the important role of intercellular interaction in organ development and homeostasis.

Genetic mutations have been reported in a number of bone disorders with or without extra-skeletal manifestations. The purpose of the present study was to investigate the genetic cause in a middle-aged woman with osteoporosis, recurrent fractures and extraskeletal manifestations.

A 56-year-old Indian woman presented to the clinic with complaints of difficulty in walking, recurrent fractures, limb bending, progressive skeletal deformities, and poor overall health. At the age of 37, she had experienced severe anemia with diarrhea, significant weight loss, knuckle pigmentation, and a significant loss of scalp hair. She had received multiple blood transfusions and parenteral iron supplementation with normalization of hemoglobin. Subsequently, she had premature menopause at the age of 37. She died at the age of 61 due to liver failure. Exome sequencing followed by Sanger sequencing were undertaken to identify the potential pathogenic mutations.

Genetic investigation identified likely pathogenic mutations in the LRP5 and LGR4 genes. Out of the two mutations, the heterozygous mutation (c.1199C>T) in the LRP5 gene resulted in a non-synonymous substitution of alanine with valine at the 400th position, and the second mutation (c.1403A>C) in the LGR4 gene led to a non-synonymous substitution of tyrosine with serine at the 468th residue of the protein. The minor allele frequencies of the c.1199C>T (LRP5) substitution in the 1000 genomes and IndiGenomes databases are 0.0003 and 0.001, while the c.1403A>C (LGR4) substitution has not been reported in these databases. Various in silico prediction tools suggested LGR4 mutation to be pathogenic and LRP5 mutation to be likely pathogenic.

Heterozygous mutations in the LRP5 and LGR4 genes had additive deteriorative effects on BMD, resulting in recurrent fractures and bone deformities, and extended the effect to extraskeletal sites, contributing to the poor overall health in this patient.

The class F of G protein-coupled receptors (GPCRs) consists of 10 Frizzleds (FZD1-10) and Smoothened (SMO). FZDs bind and are activated by secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, and SMO is indirectly activated by the Hedgehog (Hh) family of morphogens acting on the transmembrane protein Patched. The advance of our understanding of FZDs and SMO as dynamic transmembrane receptors and molecular machines, which emerged during the past 14 years since the first-class F GPCR IUPHAR nomenclature report, justifies an update. This article focuses on the advances in molecular pharmacology and structural biology providing new mechanistic insight into ligand recognition, receptor activation mechanisms, signal initiation, and signal specification. Furthermore, class F GPCRs continue to develop as drug targets, and novel technologies and tools such as genetically encoded biosensors and CRISP/Cas9 edited cell systems have contributed to refined functional analysis of these receptors. Also, advances in crystal structure analysis and cryogenic electron microscopy contribute to the rapid development of our knowledge about structure-function relationships, providing a great starting point for drug development. Despite the progress, questions and challenges remain to fully understand the complexity of the WNT/FZD and Hh/SMO signaling systems. SIGNIFICANCE STATEMENT: The recent years of research have brought about substantial functional and structural insight into mechanisms of activation of Frizzleds and Smoothened. While the advance furthers our mechanistic understanding of ligand recognition, receptor activation, signal specification, and initiation, broader opportunities emerge that allow targeting class F GPCRs for therapy and regenerative medicine employing both biologics and small molecule compounds.

Regeneration is a heroic biological process that restores tissue architecture and function in the face of day-to-day cell loss or the aftershock of injury. Capacities and mechanisms for regeneration can vary widely among species, organs, and injury contexts. Here, we describe "hallmarks" of regeneration found in diverse settings of the animal kingdom, including activation of a cell source, initiation of regenerative programs in the source, interplay with supporting cell types, and control of tissue size and function. We discuss these hallmarks with an eye toward major challenges and applications of regenerative biology.

The normal liver has an extraordinary capacity of regeneration. However, this capacity is significantly impaired in steatotic livers. Emerging evidence indicates that metabolic dysfunction associated steatotic liver disease (MASLD) and liver regeneration share several key mechanisms. Some classical liver regeneration pathways, such as HGF/c-Met, EGFR, Wnt/β-catenin and Hippo/YAP-TAZ are affected in MASLD. Some recently established therapeutic targets for MASH such as the Thyroid Hormone (TH) receptors, Glucagon-like protein 1 (GLP1), Farnesoid X receptor (FXR), Peroxisome Proliferator-Activated Receptors (PPARs) as well as Fibroblast Growth Factor 21 (FGF21) are also reported to affect hepatocyte proliferation. With this review we aim to provide insight into common molecular pathways, that may ultimately enable therapeutic strategies that synergistically ameliorate steatohepatitis and improve the regenerating capacity of steatotic livers. With the recent rise of prolonged ex-vivo normothermic liver perfusion prior to organ transplantation such treatment is no longer restricted to patients undergoing major liver resection or transplantation, but may eventually include perfused (steatotic) donor livers or even liver segments, opening hitherto unexplored therapeutic avenues.

Graft-versus-host disease (GVHD) is a major life-threatening complication that occurs after allogeneic hematopoietic cell transplantation (HCT). Although adult tissue stem cells have been identified as targets of GVHD in the skin and gut, their role in hepatic GVHD is yet to be clarified. In the current study, we explored the fate of bile duct stem cells (BDSCs), capable of generating liver organoids in vitro, during hepatic GVHD after allogeneic HCT. We observed a significant expansion of biliary epithelial cells (BECs) on injury early after allogeneic HCT. Organoid-forming efficiency from the bile duct was also significantly increased early after allogeneic HCT. Subsequently, the organoid-forming efficiency from bile ducts was markedly decreased in association with the reduction of BECs and the elevation of plasma concentrations of bilirubin, suggesting that GVHD targets BDSCs and impairs the resilience of BECs. The growth of liver organoids in the presence of liver-infiltrating mononuclear cells from allogeneic recipients, but not from syngeneic recipients, was significantly reduced in a transforming growth factor-β (TGF-β)-dependent manner. Administration of SB-431542, an inhibitor of TGF-β signaling, from day 14 to day 28, protected organoid-forming BDSCs against GVHD and mitigated biliary dysfunction after allogeneic HCT, suggesting that BDSCs are a promising therapeutic target for hepatic GVHD.

Binucleated polyploid cells are common in many animal tissues, where they arise by endomitosis, a non-canonical cell cycle in which cells enter M phase but do not undergo cytokinesis. Different steps of cytokinesis have been shown to be inhibited during endomitosis M phase in rodents, but it is currently unknown how human cells undergo endomitosis. In this study, we use fetal-derived human hepatocyte organoids (Hep-Orgs) to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, which is associated with the loss of four cortical anchoring proteins, RacGAP1, Anillin, SEPT9, and citron kinase (CIT-K). Moreover, reduction of WNT activity increases the percentage of binucleated cells in Hep-Orgs, an effect that is dependent on the atypical E2F proteins, E2F7 and E2F8. Together, we have elucidated how hepatocytes undergo endomitosis in human Hep-Orgs, providing new insights into the mechanisms of endomitosis in mammals.

Adult tissues set the scene for a continuous battle between cells, where a comparison of cellular fitness results in the elimination of weaker "loser" cells. This phenomenon, named cell competition, is beneficial for tissue integrity and homeostasis. In fact, cell competition plays a crucial role in tumor suppression, through elimination of early malignant cells, as part of Epithelial Defense Against Cancer. However, it is increasingly apparent that cell competition doubles as a tumor-promoting mechanism. The comparative nature of cell competition means that mutational background, proliferation rate and polarity all factor in to determine the outcome of these processes. In this review, we explore the intricate and context-dependent involvement of cell competition in homeostasis and regeneration, as well as during initiation and progression of primary and metastasized colorectal cancer. We provide a comprehensive overview of molecular and cellular mechanisms governing cell competition and its parallels with regeneration.

Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 1010 primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1 ± 28.1 µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems.

Wingless-related integration site or Wingless and Int-1 or Wingless-Int (WNT) signaling is crucial for embryonic development, and adult tissue homeostasis and regeneration, through its essential roles in cell fate, patterning, and stem cell regulation. The biophysical characteristics of WNT ligands have hindered efforts to interrogate ligand activity in vivo and prevented their development as therapeutics. Recent breakthroughs have enabled the generation of synthetic WNT signaling molecules that possess characteristics of natural ligands and potently activate the pathway, while also providing distinct advantages for therapeutic development and manufacturing. This review provides a detailed discussion of the protein engineering of these molecular platforms for WNT signaling agonism. We discuss the importance of WNT signaling in several organs and share insights from the initial application of these new classes of molecules in vitro and in vivo. These molecules offer a unique opportunity to enhance our understanding of how WNT signaling agonism promotes tissue repair, enabling targeted development of tailored therapeutics.

Recent advancements in genome editing techniques, notably CRISPR-Cas9 and TALENs, have marked a transformative era in biomedical research, significantly enhancing our understanding of disease mechanisms and helping develop novel therapies. These technologies have been instrumental in creating precise animal models for use in stem cell research and regenerative medicine. For instance, we have developed a transgenic pig model to enable the investigation of LGR5-expressing cells. The model was designed to induce the expression of H2B-GFP under the regulatory control of the LGR5 promoter via CRISPR/Cas9-mediated gene knock-in. Notably, advancements in stem cell research have identified distinct subpopulations of LGR5-expressing cells within adult human, mouse, and pig tissues. LGR5, a leucine-rich repeat-containing G protein-coupled receptor, enhances WNT signaling and these LGR5+ subpopulations demonstrate varied roles and anatomical distributions, underscoring the necessity for suitable translational models. This transgenic pig model facilitates the tracking of LGR5-expressing cells and has provided valuable insights into the roles of these cells across different tissues and species. For instance, in pulmonary tissue, Lgr5+ cells in mice are predominantly located in alveolar compartments, driving alveolar differentiation of epithelial progenitors via Wnt pathway activation. In contrast, in pigs and humans, these cells are situated in a unique sub-basal position adjacent to the airway epithelium. In fetal stages a pattern of LGR5 expression during lung bud tip formation is evident in humans and pigs but is lacking in mice. Species differences with respect to LGR5 expression have also been observed in the skin, intestines, and cochlea further reinforcing the need for careful selection of appropriate translational animal models. This paper discusses the potential utility of the LGR5+ pig model in exploring the role of LGR5+ cells in tissue development and regeneration with the goal of translating these findings into human and animal clinical applications.

Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal hepatic function. Wnt signaling is a master regulator of spatial liver zonation. A perivenous-periportal Wnt activity gradient orchestrates metabolic zonation by activating gene expression in perivenous hepatocytes, while suppressing gene expression in their periportal counterparts. However, the understanding as to the liver gene zonation and zonation regulators in diseases is limited. Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fat accumulation, inflammation, and fibrosis. Here, we investigated the perturbation of liver gene zonation in a mouse NASH model by combining spatial transcriptomics, bulk RNAseq and in situ hybridization. Wnt-target genes represented a major subset of genes showing altered spatial expression in the NASH liver. The altered Wnt-target gene expression levels and zonation spatial patterns were in line with the up regulation of Wnt regulators and the augmentation of Wnt signaling. Particularly, we found that the Wnt activator Rspo3 expression was restricted to the perivenous zone in control liver but expanded to the periportal zone in NASH liver. AAV8-mediated RSPO3 overexpression in controls resulted in zonation changes, and further amplified the disturbed zonation of Wnt-target genes in NASH, similarly Rspo3 knockdown in Rspo3+/- mice resulted in zonation changes of Wnt-target genes in both chow and HFD mouse. Interestingly, there were no impacts on steatosis, inflammation, or fibrosis NASH pathology from RSPO3 overexpression nor Rspo3 knockdown. In summary, our study demonstrated the alteration of Wnt signaling in a mouse NASH model, leading to perturbed liver zonation.

The Wnt/β-catenin pathway is an evolutionarily conserved signaling pathway that controls fundamental physiological and pathological processes by regulating cell proliferation and differentiation. The Wnt/β-catenin pathway enables liver homeostasis by inducing differentiation and contributes to liver-specific features such as metabolic zonation and regeneration. In contrast, abnormalities in the Wnt/β-catenin pathway promote the development and progression of hepatocellular carcinoma (HCC). Similarly, hepatoblastoma, the most common childhood liver cancer, is frequently associated with β-catenin mutations, which activate Wnt/β-catenin signaling. HCCs with activation of the Wnt/β-catenin pathway have unique gene expression patterns and pathological and clinical features. Accordingly, they are highly differentiated with retaining hepatocyte-like characteristics and tumorigenic. Activation of the Wnt/β-catenin pathway in HCC also alters the state of immune cells, causing "immune evasion" with inducing resistance to immune checkpoint inhibitors, which have recently become widely used to treat HCC. Activated Wnt/β-catenin signaling exhibits these phenomena in liver tumorigenesis through the expression of downstream target genes, and the molecular basis is still poorly understood. In this review, we describe the physiological roles of Wnt/b-catenin signaling and then discuss their characteristic changes by the abnormal activation of Wnt/b-catenin signaling. Clarification of the mechanism would contribute to the development of therapeutic agents in the future.

The liver acts as a master regulator of metabolic homeostasis in part by performing gluconeogenesis. This process is dysregulated in type 2 diabetes, leading to elevated hepatic glucose output. The parenchymal cells of the liver (hepatocytes) are heterogeneous, existing on an axis between the portal triad and the central vein, and perform distinct functions depending on location in the lobule. Here, using single cell analysis of hepatocytes across the liver lobule, we demonstrate that gluconeogenic gene expression ( Pck1 and G6pc ) is relatively low in the fed state and gradually increases first in the periportal hepatocytes during the initial fasting period. As the time of fasting progresses, pericentral hepatocyte gluconeogenic gene expression increases, and following entry into the starvation state, the pericentral hepatocytes show similar gluconeogenic gene expression to the periportal hepatocytes. Similarly, pyruvate-dependent gluconeogenic activity is approximately 10-fold higher in the periportal hepatocytes during the initial fasting state but only 1.5-fold higher in the starvation state. In parallel, starvation suppresses canonical beta-catenin signaling and modulates expression of pericentral and periportal glutamine synthetase and glutaminase, resulting in an enhanced pericentral glutamine-dependent gluconeogenesis. These findings demonstrate that hepatocyte gluconeogenic gene expression and gluconeogenic activity are highly spatially and temporally plastic across the liver lobule, underscoring the critical importance of using well-defined feeding and fasting conditions to define the basis of hepatic insulin resistance and glucose production.

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor and one of the leading causes of cancer-related deaths worldwide. The Wnt/β-Catenin signaling pathway is a highly conserved pathway involved in several biological processes, including the improper regulation that leads to the tumorigenesis and progression of cancer. New studies have found that abnormal activation of the Wnt/β-Catenin signaling pathway is a major cause of HCC tumorigenesis, progression, and resistance to therapy. New perspectives and approaches to treating HCC will arise from understanding this pathway. This article offers a thorough analysis of the Wnt/β-Catenin signaling pathway's function and its therapeutic implications in HCC.

The leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) plays an important role in stem cell differentiation, organ development and cancer. Whether LGR4 affects the progression of hepatocellular carcinoma (HCC) remains unknown. This study aimed to reveal the role of LGR4 in HCC.

Clinical samples of HCC were collected to assess the expression of LGR4 and its correlation with patients' clinical characteristics. The expression level of LGR4 in HCC cells was altered by pharmacological and genetic methods, and the role of LGR4 in HCC progression was analyzed by in vivo and in vitro assays. HCC was induced by diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) in wild-type and LGR4 deficient mice, the effect of LGR4 on HCC was examined by histopathological evaluation and biochemical assays.

LGR4 expression was up-regulated in HCC samples, and its expression level was positively correlated with tumor size, microvascular invasion (MVI), TNM stage and pathological differentiation grade of HCC patients. In the mouse HCC model induced by DEN+CCl4, knockdown of LGR4 effectively inhibited the progression of HCC. Silencing of LGR4 inhibited the proliferation, migration, invasion, stem cell-like properties and Warburg effect of HCC cells. These phenotypes were promoted by R-spondin2 (Rspo2), an endogenous ligand for LGR4. Rspo2 markedly increased the nuclear translocation of β-catenin, whereas IWR-1, an inhibitor of Wnt/β-catenin signaling, reversed its effect. Deficiency of LGR4 significantly reduced the nuclear translocation of β-catenin and the expression of its downstream target genes cyclinD1 and c-Myc.

LGR4 promotes HCC progression via Wnt/β-catenin signaling pathway.

The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/β-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program.

Liver injuries often occur in a zonated manner. However, detailed regenerative responses to such zonal injuries at cellular and molecular levels remain largely elusive. By using a fate-mapping strain, Cyp2e1-DreER, to elucidate liver regeneration after acute pericentral injury, we found that pericentral regeneration is primarily compensated by the expansion of remaining pericentral hepatocytes, and secondarily by expansion of periportal hepatocytes. Employing single-cell RNA sequencing, spatial transcriptomics, immunostaining, and in vivo functional assays, we demonstrated that the upregulated expression of the mTOR/4E-BP1 axis and lactate dehydrogenase A in hepatocytes contributes to pericentral regeneration, while activation of transforming growth factor β (TGF-β1) signaling in the damaged area mediates fibrotic responses and inhibits hepatocyte proliferation. Inhibiting the pericentral accumulation of monocytes and monocyte-derived macrophages through an Arg-Gly-Asp (RGD) peptide-based strategy attenuates these cell-derived TGF-β1 signalings, thus improving pericentral regeneration. Our study provides integrated and high-resolution spatiotemporal insights into the cellular and molecular basis of pericentral regeneration.