Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Opposing roles of hepatic stellate cell subpopulations in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis1,2. Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts3, during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion.

Lipid droplets are intracellular mechanical stressors that impair hepatocyte function.

Matrix stiffening and external mechanical stress have been linked to disease and cancer development in multiple tissues, including the liver, where cirrhosis (which increases stiffness markedly) is the major risk factor for hepatocellular carcinoma. Patients with nonalcoholic fatty liver disease and lipid droplet-filled hepatocytes, however, can develop cancer in noncirrhotic, relatively soft tissue. Here, by treating primary human hepatocytes with the monounsaturated fatty acid oleate, we show that lipid droplets are intracellular mechanical stressors with similar effects to tissue stiffening, including nuclear deformation, chromatin condensation, and impaired hepatocyte function. Mathematical modeling of lipid droplets as inclusions that have only mechanical interactions with other cellular components generated results consistent with our experiments. These data show that lipid droplets are intracellular sources of mechanical stress and suggest that nuclear membrane tension integrates cell responses to combined internal and external stresses.

Glycosaminoglycans modulate long-range mechanical communication between cells in collagen networks.

Cells can sense and respond to mechanical forces in fibrous extracellular matrices (ECMs) over distances much greater than their size. This phenomenon, termed long-range force transmission, is enabled by the realignment (buckling) of collagen fibers along directions where the forces are tensile (compressive). However, whether other key structural components of the ECM, in particular glycosaminoglycans (GAGs), can affect the efficiency of cellular force transmission remains unclear. Here we developed a theoretical model of force transmission in collagen networks with interpenetrating GAGs, capturing the competition between tension-driven collagen fiber alignment and the swelling pressure induced by GAGs. Using this model, we show that the swelling pressure provided by GAGs increases the stiffness of the collagen network by stretching the fibers in an isotropic manner. We found that the GAG-induced swelling pressure can help collagen fibers resist buckling as the cells exert contractile forces. This mechanism impedes the alignment of collagen fibers and decreases long-range cellular mechanical communication. We experimentally validated the theoretical predictions by comparing the intensity of collagen fiber alignment between cellular spheroids cultured on collagen gels versus collagen­GAG cogels. We found significantly lower intensities of aligned collagen in collagen­GAG cogels, consistent with the prediction that GAGs can prevent collagen fiber alignment. The role of GAGs in modulating force transmission uncovered in this work can be extended to understand pathological processes such as the formation of fibrotic scars and cancer metastasis, where cells communicate in the presence of abnormally high concentrations of GAGs.

Biliary atresia: insights into mechanisms using a toxic model of the disease including Wnt and Hippo signaling pathways and microtubules.

BACKGROUND: Mechanisms underlying bile duct injury in biliary atresia (BA) remain unclear and mechanisms of bile duct repair are unknown. This study aimed to explore the roles of microtubule instability and Wnt and Hippo signaling pathways in a biliatresone-induced BA model. METHODS: Using primary murine neonatal cholangiocytes in both 2D and 3D cultures, and ex-vivo extra hepatic bile ducts (EHBD) which also has peri-cholangiocyte area, we analyzed injury and recovery processes. Injury was induced by the toxin biliatresone and recovery was induced by toxin wash-out. RESULTS: Microtubule stabilizer paclitaxel prevented biliatresone-induced injury, both to cholangiocytes as well as reduced periductal αSMA stain, this process is mediated by decreased glutathione levels. RhoU and Wnt11 (Wnt signaling) and Pard6g and Amotl1 (Hippo signaling) are involved in both injury and recovery processes, with the latter acting upstream to Wnt signaling. CONCLUSIONS: Early stages of biliatresone-induced EHBD injury in cholangiocytes and periductal structures are reversible. Wnt and Hippo signaling pathways play crucial roles in injury and recovery, providing insights into BA injury mechanisms and potential recovery avenues. IMPACT: Microtubule stabilization prevents cholangiocyte injury and lumen obstruction in a toxic model of biliary atresia (biliatresone induced). Early stages of biliatresone-induced injury, affecting both cholangiocytes and periductal structures, are reversible. Both Wnt and Hippo signaling pathways play a crucial role in bile duct injury and recovery, with a noted interplay between the two. Understanding mechanisms of cholangiocyte recovery is imperative to unveil potential therapeutic avenues.

In Utero Extrahepatic Bile Duct Damage and Repair: Implications for Biliary Atresia.

Biliary atresia (BA) is a cholangiopathy affecting the extrahepatic bile duct (EHBD) of newborns. The etiology and pathophysiology of BA are not fully understood; however, multiple causes of damage and obstruction of the neonatal EHBD have been identified. Initial damage to the EHBD likely occurs before birth. We discuss how different developmental stages in utero and birth itself could influence the susceptibility of the fetal EHBD to damage and a damaging wound-healing response. We propose that a damage-repair response of the fetal and neonatal EHBD involving redox stress and a program of fetal wound healing could-regardless of the cause of the initial damage-lead to either obstruction and BA or repair of the duct and recovery. This overarching concept should guide future research targeted toward identification of factors that contribute to recovery as opposed to progression of injury and fibrosis. Viewing BA through the lens of an in utero damage-repair response could open up new avenues for research and suggests exciting new therapeutic targets.

A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

Conjugated hyperbilirubinemia is associated with increased morbidity and mortality after neonatal heart surgery.

BACKGROUND: Cholestasis characterised by conjugated hyperbilirubinemia is a marker of hepatobiliary dysfunction following neonatal cardiac surgery. We aimed to characterise the incidence of conjugated hyperbilirubinemia following neonatal heart surgery and examine the effect of conjugated hyperbilirubinemia on post-operative morbidity and mortality. METHODS: This was a retrospective study of all neonates who underwent surgery for congenital heart disease (CHD) at our institution between 1/1/2010 and 12/31/2020. Patient- and surgery-specific data were abstracted from local registry data and review of the medical record. Conjugated hyperbilirubinemia was defined as perioperative maximum conjugated bilirubin level > 1 mg/dL. The primary outcome was in-hospital mortality. Survival analysis was conducted using the Kaplan-Meier survival function. RESULTS: Conjugated hyperbilirubinemia occurred in 8.5% of patients during the study period. Neonates with conjugated hyperbilirubinemia were more likely to be of younger gestational age, lower birth weight, and non-Caucasian race (all p < 0.001). Patients with conjugated hyperbilirubinemia were more likely to have chromosomal and non-cardiac anomalies and require ECMO pre-operatively. In-hospital mortality among patients with conjugated hyperbilirubinemia was increased compared to those without (odds ratio 5.4). Post-operative complications including mechanical circulatory support, reoperation, prolonged ventilator dependence, and multi-system organ failure were more common with conjugated hyperbilirubinemia (all p < 0.04). Patients with higher levels of conjugated bilirubin had worst intermediate-term survival, with patients in the highest conjugated bilirubin group (>10 mg/dL) having a 1-year survival of only 6%. CONCLUSIONS: Conjugated hyperbilirubinemia is associated with post-operative complications and worse survival following neonatal heart surgery. Cholestasis is more common in patients with chromosomal abnormalities and non-cardiac anomalies, but the underlying mechanisms have not been delineated.

What's old is new again: The anatomical studies of Franklin P. Mall and the fascial-interstitial spaces.

Franklin Mall was one of the foremost scientists of the turn of the 19th century, an exemplary mentor as well as researcher, and his revolutionary contributions are still relevant today. Mall's early training in Leipzig with Wilhelm His and Carl Ludwig provided him with an unusual perspective on the integration of anatomy and physiology, and his interest in the links between structure and function guided the work he carried out after joining the faculty of the new Johns Hopkins University School of Medicine. Mall carried out innovative studies on the one hand using dye injection to trace blood and lymphatic supplies to different organs and on the other hand using "putrefaction" to digest tissues and study the organization of the reticular space, demonstrating that it was the underlying source of support for all the organs. These two studies of Mall's, carried out independently, provide the basis for modern studies integrating the understanding of fascia and interstitial spaces.

A Bile Duct-on-a-Chip With Organ-Level Functions.

BACKGROUND AND AIMS: Chronic cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), are frequently associated with damage to the barrier function of the biliary epithelium. Here, we report on a bile duct-on-a-chip that phenocopies not only the tubular architecture of the bile duct in three dimensions, but also its barrier functions. APPROACH AND RESULTS: We showed that mouse cholangiocytes in the channel of the device became polarized and formed mature tight junctions, that the permeability of the cholangiocyte monolayer was comparable to ex vivo measurements, and that cholangiocytes in the device were mechanosensitive (as demonstrated by changes in calcium flux under applied luminal flow). Permeability decreased significantly when cells formed a compact monolayer with cell densities comparable to those observed in vivo. This device enabled independent access to the apical and basolateral surfaces of the cholangiocyte channel, allowing proof-of-concept toxicity studies with the biliary toxin, biliatresone, and the bile acid, glycochenodeoxycholic acid. The cholangiocyte basolateral side was more vulnerable than the apical side to treatment with either agent, suggesting a protective adaptation of the apical surface that is normally exposed to bile. Further studies revealed a protective role of the cholangiocyte apical glycocalyx, wherein disruption of the glycocalyx with neuraminidase increased the permeability of the cholangiocyte monolayer after treatment with glycochenodeoxycholic acid. CONCLUSIONS: This bile duct-on-a-chip captured essential features of a simplified bile duct in structure and organ-level functions and represents an in vitro platform to study the pathophysiology of the bile duct using cholangiocytes from a variety of sources.

A genome-wide association study identifies a susceptibility locus for biliary atresia on 2p16.1 within the gene EFEMP1.

Biliary atresia (BA) is a rare pediatric cholangiopathy characterized by fibrosclerosing obliteration of the extrahepatic bile ducts, leading to cholestasis, fibrosis, cirrhosis, and eventual liver failure. The etiology of BA remains unknown, although environmental, inflammatory, infectious, and genetic risk factors have been proposed. We performed a genome-wide association study (GWAS) in a European-American cohort of 343 isolated BA patients and 1716 controls to identify genetic loci associated with BA. A second GWAS was performed in an independent European-American cohort of 156 patients with BA and other extrahepatic anomalies and 212 controls to confirm the identified candidate BA-associated SNPs. Meta-analysis revealed three genome-wide significant BA-associated SNPs on 2p16.1 (rs10865291, rs6761893, and rs727878; P < 5 ×10-8), located within the fifth intron of the EFEMP1 gene, which encodes a secreted extracellular protein implicated in extracellular matrix remodeling, cell proliferation, and organogenesis. RNA expression analysis showed an increase in EFEMP1 transcripts from human liver specimens isolated from patients with either BA or other cholestatic diseases when compared to normal control liver samples. Immunohistochemistry demonstrated that EFEMP1 is expressed in cholangiocytes and vascular smooth muscle cells in liver specimens from patients with BA and other cholestatic diseases, but it is absent from cholangiocytes in normal control liver samples. Efemp1 transcripts had higher expression in cholangiocytes and portal fibroblasts as compared with other cell types in normal rat liver. The identification of a novel BA-associated locus, and implication of EFEMP1 as a new BA candidate susceptibility gene, could provide new insights to understanding the mechanisms underlying this severe pediatric disorder.

Robust cellular reprogramming occurs spontaneously during liver regeneration.

Cellular reprogramming-the ability to interconvert distinct cell types with defined factors-is transforming the field of regenerative medicine. However, this phenomenon has rarely been observed in vivo without exogenous factors. Here, we report that activation of Notch, a signaling pathway that mediates lineage segregation during liver development, is sufficient to reprogram hepatocytes into biliary epithelial cells (BECs). Moreover, using lineage tracing, we show that hepatocytes undergo widespread hepatocyte-to-BEC reprogramming following injuries that provoke a biliary response, a process requiring Notch. These results provide direct evidence that mammalian regeneration prompts extensive and dramatic changes in cellular identity under injury conditions.

Coordinated development of the mouse extrahepatic bile duct: Implications for neonatal susceptibility to biliary injury.

BACKGROUND & AIMS: The extrahepatic bile duct is the primary tissue initially affected by biliary atresia. Biliary atresia is a cholangiopathy which exclusively affects neonates. Current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts. METHODS: We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes, as well as isolated extrahepatic bile ducts, and a collagen reporter mouse. The methods used included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix. RESULTS: The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling of the matrix suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. CONCLUSION: We identified 4 key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis. LAY SUMMARY: Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction, resulting in liver injury. We identify 4 key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.

Lipid droplets disrupt mechanosensing in human hepatocytes.

Hepatocellular carcinoma (HCC) is the fourth-leading cause of cancer death in the world. Although most cases occur in stiff, cirrhotic livers, and stiffness is a significant risk factor, HCC can also arise in noncirrhotic livers in the setting of nonalcoholic fatty liver disease (NAFLD). We hypothesized that lipid droplets in NAFLD might apply mechanical forces to the nucleus, functioning as mechanical stressors akin to stiffness. We investigated the effect of lipid droplets on cellular mechanosensing and found that primary human hepatocytes loaded with the fatty acids oleate and linoleate exhibited decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers. The presence of large lipid droplets in hepatocytes resulted in increased nuclear localization of the mechano-sensor Yes-associated protein (YAP). In cirrhotic livers from patients with NAFLD, hepatocytes filled with large lipid droplets showed significantly higher nuclear localization of YAP as compared with cells with small lipid droplets. This work suggests that lipid droplets induce a mechanical signal that disrupts the ability of the hepatocyte to sense its underlying matrix stiffness and that the presence of lipid droplets can induce intracellular mechanical stresses.NEW & NOTEWORTHY This work examines the impact of lipid loading on mechanosensing by human hepatocytes. In cirrhotic livers, the presence of large (although not small) lipid droplets increased nuclear localization of the mechanotransducer YAP. In primary hepatocytes in culture, lipid droplets led to decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers; the presence of large lipid droplets resulted in increased YAP nuclear localization. Collectively, the data suggest that lipid droplets induce intracellular mechanical stress.

The fibronectin ED-A domain enhances recruitment of latent TGF-ß-binding protein-1 to the fibroblast matrix.

Dysregulated secretion and extracellular activation of TGF-ß1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) leading to fibrosis. Fibronectin immobilizes latent TGF-ß-binding protein-1 (LTBP-1) and thus stores TGF-ß1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed during fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1-fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and colocalization of LTBP-1 and ED-A-containing fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A-containing fibronectin than to ED-B-containing fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-A-containing fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM and reduced TGF-ß1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-ß1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin.

Liver Cancer Gene Discovery Using Gene Targeting, Sleeping Beauty, and CRISPR/Cas9.

Hepatocellular carcinoma (HCC) is a devastating and prevalent cancer with limited treatment options. Technological advances have enabled genetic screens to be employed in HCC model systems to characterize genes regulating tumor initiation and growth. Relative to traditional methods for studying cancer biology, such as candidate gene approaches or expression analysis, genetic screens have several advantages: they are unbiased, with no a priori selection; can directly annotate gene function; and can uncover gene-gene interactions. In HCC, three main types of screens have been conducted and are reviewed here: (1) transposon-based mutagenesis screens, (2) knockdown screens using RNA interference (RNAi) or the CRISPR/Cas9 system, and (3) overexpression screens using CRISPR activation (CRISPRa) or cDNAs. These methods will be valuable in future genetic screens to delineate the mechanisms underlying drug resistance and to identify new treatments for HCC.

Biliary Atresia: Clinical and Research Challenges for the Twenty-First Century.

Biliary atresia (BA) is a fibroinflammatory disease of the intrahepatic and extrahepatic biliary tree. Surgical hepatic portoenterostomy (HPE) may restore bile drainage, but progression of the intrahepatic disease results in complications of portal hypertension and advanced cirrhosis in most children. Recognizing that further progress in the field is unlikely without a better understanding of the underlying cause(s) and pathogenesis of the disease, the National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored a research workshop focused on innovative and promising approaches and on identifying future areas of research. Investigators discussed recent advances using gestational ultrasound and results of newborn BA screening with serum direct (conjugated) bilirubin that support a prenatal onset of biliary injury. Experimental and human studies implicate the toxic properties of environmental toxins (e.g., biliatresone) and of viruses (e.g., cytomegalovirus) to the biliary system. Among host factors, sequence variants in genes related to biliary development and ciliopathies, a notable lack of a cholangiocyte glycocalyx and of submucosal collagen bundles in the neonatal extrahepatic bile ducts, and an innate proinflammatory bias of the neonatal immune system contribute to an increased susceptibility to damage and obstruction following epithelial injury. These advances form the foundation for a future research agenda focused on identifying the environmental and host factor(s) that cause BA, the potential use of population screening, studies of the mechanisms of prominent fibrosis in young infants, determinations of clinical surrogates of disease progression, and the design of clinical trials that target subgroups of patients with initial drainage following HPE. (Hepatology 2018; 00:000-000).

Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential.

Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1(+) cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1(+) cells had proliferative potential. Foxl1(+) cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1(+) cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

Mechanisms of Plastic Deformation in Collagen Networks Induced by Cellular Forces.

Contractile cells can reorganize fibrous extracellular matrices and form dense tracts of fibers between neighboring cells. These tracts guide the development of tubular tissue structures and provide paths for the invasion of cancer cells. Here, we studied the mechanisms of the mechanical plasticity of collagen tracts formed by contractile premalignant acinar cells and fibroblasts. Using fluorescence microscopy and second harmonic generation, we quantified the collagen densification, fiber alignment, and strains that remain within the tracts after cellular forces are abolished. We explained these observations using a theoretical fiber network model that accounts for the stretch-dependent formation of weak cross-links between nearby fibers. We tested the predictions of our model using shear rheology experiments. Both our model and rheological experiments demonstrated that increasing collagen concentration leads to substantial increases in plasticity. We also considered the effect of permanent elongation of fibers on network plasticity and derived a phase diagram that classifies the dominant mechanisms of plasticity based on the rate and magnitude of deformation and the mechanical properties of individual fibers. Plasticity is caused by the formation of new cross-links if moderate strains are applied at small rates or due to permanent fiber elongation if large strains are applied over short periods. Finally, we developed a coarse-grained model for plastic deformation of collagen networks that can be employed to simulate multicellular interactions in processes such as morphogenesis, cancer invasion, and fibrosis.