Induction of angiogenesis and neural progenitor cells by basic fibroblast growth factor-releasing polyglycolic acid sheet following focal cerebral infarction in mice.

Biodegradable sheets loaded with basic fibroblast growth factor (bFGF) are prepared as novel bFGF-releasing systems from polyglycolic acid nonwoven fabric by oxygen plasma treatment followed by bFGF adsorption. In the present study, we investigated the therapeutic effects of this system on a focal cerebral infarction model (CB-17 mouse). A preliminary in vitro study showed that this system released bFGF in an acellular culture medium, thereby keeping the bFGF concentration in the medium at ≥5 ng/ml for a prolonged period of 7 days. The released bFGF from this system retained its biological activity to enhance endothelial tube formation in vitro. In a mouse model of subacute focal cerebral infarction, this system increased the expression of endogenous vascular endothelial growth factor in the peri-infarct cortex and subventricular zone, promoted angiogenesis in the striatum, and increased neural progenitor cells in the peri-infarct cortex. Thus, this bFGF-releasing system has the potential to be a novel therapeutic approach for cerebral infarction.

The enhancing effects of heparin on the biological activity of FGF-2 in heparin-FGF-2-calcium phosphate composite layers.

Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the composite layers is important for its wider application in orthopedics and dentistry. This study incorporated low-molecular-weight heparin (LMWH) into the FGF-2-calcium phosphate composite layers and clarified the enhancing effects of LMWH on the biological activity of FGF-2 in the composite layers in vitro. LMWH-FGF-2-calcium phosphate composite layers were successfully formed on zirconia in supersaturated calcium phosphate solutions. The composite layers comprised continuous and macroscopically homogeneous layers and particles smaller than 500 nm in size composed of amorphous calcium phosphate. The amounts of Ca and P deposited on zirconia remained almost unchanged with the addition of LMWH under the presence of FGF-2 in the supersaturated calcium phosphate solution. The LMWH in the supersaturated calcium phosphate solution increased the stability of FGF-2 in the solution and the amount of FGF-2 in the composite layers. The LMWH in the composite layers increased the mitogenic and endothelial tube-forming activities of FGF-2, and FGF-2 activity of inducing osteogenic differentiation gene expression pattern in the composite layers. Our results indicate that the enhanced biological activity of FGF-2 in the LMWH-FGF-2-calcium phosphate composite layers is attributed to an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the supersaturated calcium phosphate solution and the composite layers. The LMWH-FGF-2-calcium phosphate composite layer is a promising coating for orthopedic and dental implants. STATEMENT OF SIGNIFICANCE: Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the layers is important for wider its application in orthopedics and dentistry. This study demonstrates the enhancing effects of low-molecular-weight heparin (LMWH) contained within LMWH-FGF-2-calcium phosphate composite layers on the biological activity of FGF-2 in vitro. Our results indicate that the enhanced biological activity of FGF-2 within the composite layers arises from an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the LMWH-FGF-2-calcium phosphate composite layers and supersaturated calcium phosphate solutions used for coating the composite layers.

Iron-accumulating splenocytes may exacerbate non-alcoholic steatohepatitis through the production of proinflammatory cytokines and reactive oxygen species.

Non-alcoholic steatohepatitis (NASH) results from non-alcoholic fatty liver disease (NAFLD) via multiple-parallel events, including hepatic triglyceride accumulation, oxidative stress, and inflammation. The complex interaction between the liver and multiple other organs is involved in NASH development. Although spleen-derived humoral factors can directly contribute to NAFLD/NASH onset via the portal vein, the status of the spleen in the early stage of NASH remains unknown. Here, our aim was to investigate whether splenocytes may exacerbate NASH via the generations of reactive oxygen species (ROS) and proinflammatory cytokines. Iron accumulation was observed in the spleen but not the liver, and the proportion of phagocytic macrophages increased in the spleen of Tsumura Suzuki Obese Diabetes (TSOD) mice showing histological characteristics of NASH in the early stage. The splenocytes generated moderate amounts of ROS and released high amounts of tumor necrosis factor (TNF)-α in response to lipopolysaccharide, indicating excessive inflammatory cytokine released by activated macrophages in iron-accumulating spleens. Our results suggest that iron-accumulating splenocytes can easily induce inflammation and contribute to exacerbate NASH via the portal vein. Thus, the regulation of iron metabolism in the spleen should be considered in the development of novel therapeutic targets against NASH.

Cell attachment area of rat mesenchymal stem cells correlates with their osteogenic differentiation level on substrates without osteoconductive property.

Cell morphology is related to proliferation and differentiation. We previously reported that cell attachment area of rat mesenchymal stem cells (MSCs) is negatively correlated with their osteogenic differentiation level on osteoconductive hydroxyapatite (HAp) with various microstructures. In this study, the correlation between the cell attachment area and osteogenic differentiation level was investigated on substrates without osteoconductive property using tissue culture polystyrene (TCPS), and 3 mol% yttria-stabilized zirconia (3Y-TZP) with or without surface periodic microstructures. It was found that the osteogenic differentiation level after 3 weeks of culture increased with a decrease in cell attachment area after 3 h of culture. The square of the correlation coefficient between cell attachment area and osteocalcin secretion content was 0.845 among the three types of substrates. Thus, the negative correlation between cell attachment area and differentiation level is confirmed even when cultured on substrates without osteoconductive property. These findings suggest that the correlation between the cell attachment area of rat MSCs and osteogenic differentiation level could also apply to various types of substrate, regardless of osteoconductive property.

Function Control of Anti-microRNA Oligonucleotides Using Interstrand Cross-Linked Duplexes.

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3' UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2'-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5' and 3' termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3'-side CL improves nuclease resistance, whereas the 5'-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3' part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO.

Yuzu (Citrus junos Tanaka) Peel Attenuates Dextran Sulfate Sodium-induced Murine Experimental Colitis.

Ulcerative colitis is a well-known inflammatory bowel disease. Although there are drugs that are effective against this disease, the prevention and attenuation of ulcerative colitis by food rich in functional ingredients without side effects is desired because some drugs have side effects. In this study, we investigated the effects of yuzu (Citrus junos Tanaka), a citrus fruit native to northeast Asia, on a mouse dextran sulfate sodium (DSS)-induced colitis model. Mice given drinking water containing DSS showed significant weight loss, colon shortening, diarrhea, and visible fecal blood. In contrast, mice fed a diet containing 5% yuzu peel for 14 d before receiving DSS showed significant attenuation of these phenotypes. To clarify the mechanism underlying the attenuation, we investigated the anti-inflammatory and antioxidant effects of yuzu peel. We found that yuzu peel extract suppressed tumor necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS)-stimulated mice and murine macrophage cell line through suppression of nuclear factor-κB (NF-κB) activation. In addition, we confirmed that yuzu peel extract had a moderate antioxidant effect. These results suggest that yuzu peel attenuates the pathologies of DSS-induced colitis by coordinately suppressing inflammation and oxidative stress against lipids in vivo.

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement.

BACKGROUND: Three-dimensional (3D) spheroids are frequently used in toxicological study because their morphology and function closely resemble those of tissue. As these properties are maintained over a long term, repeated treatment of the spheroids with a test object is possible. Generally, in the repeated treatment test to assess cytotoxicity in the spheroids, ATP assay, colorimetric measurement using pigments or high-content imaging analysis is performed. However, continuous assessment of cytotoxicity in the same spheroids using the above assays or analysis is impossible because the spheroids must be disrupted or killed. To overcome this technical limitation, we constructed a simple monitoring system in which cytotoxicity in the spheroids can be continuously monitored by nondestructive bioluminescence measurement. RESULTS: Mouse primary hepatocytes were isolated from transchromosomic (Tc) mice harboring a mouse artificial chromosome (MAC) vector expressing beetle luciferase Emerald Luc (ELuc) under the control of cytomegalovirus immediate early enhancer/chicken ß-actin promoter/rabbit ß-globin intron II (CAG) promoter, and used in 3D cultures. We confirmed that both luminescence and albumin secretion from the spheroids seeded in the 96-well format Cell-ableTM were maintained for approximately 1 month. Finally, we repetitively treated the luminescent 3D spheroids with representative hepatotoxicants for approximately 1 month, and continuously and nondestructively measured bioluminescence every day. We successfully obtained daily changes of the dose-response bioluminescence curves for the respective toxicants. CONCLUSIONS: In this study, we constructed a monitoring system in which cytotoxicity in the same 3D spheroids was continuously and sensitively monitored over a long term. Because this system can be easily applied to other cells, such as human primary cells or stem cells, it is expected to serve as the preferred platform for simple and cost-effective long-term monitoring of cellular events, including cytotoxicity.

Enhanced in-cell folding of reversibly cationized transcription factor using amphipathic peptide.

The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization (S-cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAL4-VP16. Although the cationic moiety of S-cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S-cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions.

Wnt/ß-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development.

The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear. The Wnt/ß-catenin signaling pathway is involved in fundamental aspects of development through the regulation of various growth factors. Here, we show that conditional ablation of either ß-catenin (Ctnnb1(cKO)) or co-receptors Lrp5/6 (Lrp5/6(cKO)) in corneal stromal cells results in precocious stratification of the corneal epithelium. By contrast, ectopic expression of a murine Ctnnb1 gain-of-function mutant (Ctnnb1(cGOF)) retards corneal epithelium stratification. We also discovered that Bmp4 is upregulated in the absence of ß-catenin in keratocytes, which further triggers ERK1/2 (Mapk3/1) and Smad1/5 phosphorylation and enhances transcription factor p63 (Trp63) expression in mouse corneal basal epithelial cells and in a human corneal epithelial cell line (HTCE). Interestingly, mouse neonates given a subconjunctival BMP4 injection displayed a phenotype resembling that of Ctnnb1(cKO). Conditional ablation of Bmp4 eradicates the phenotype produced in Ctnnb1(cKO) mice. Furthermore, ChIP and promoter-luciferase assays show that ß-catenin binds to and suppresses Bmp4 promoter activity. These data support the concept that cross-talk between the Wnt/ß-catenin/Bmp4 axis (in the stromal mesenchyme) and Bmp4/p63 signaling (in the epithelium) plays a pivotal role in epithelial stratification during corneal morphogenesis.

Oleuropein-Rich Diet Attenuates Hyperglycemia and Impaired Glucose Tolerance in Type 2 Diabetes Model Mouse.

Oleuropein, a phenolic compound found in abundance in olive leaves, has beneficial effects on various diseases. However, it is unknown whether an oleuropein-rich diet is efficacious against type 2 diabetic phenotypes. In this study, we investigated the effects of the oleuropein-containing supplement OPIACE, whose oleuropein content exceeds 35% (w/w), on the diabetic phenotypes in type 2 diabetes model Tsumura Suzuki Obese Diabetes (TSOD) mouse. TSOD mice were fed OPIACE at 4 weeks of age, i.e., before the TSOD mice exhibited diabetic phenotypes. We revealed that OPIACE attenuated hyperglycemia and impaired glucose tolerance in TSOD mice over the long-term (from 10 to 24 weeks of age) but had no effect on obesity. Furthermore, we demonstrated that OPIACE mildly reduced oxidative stress in TSOD mice by 26.2% and attenuated anxiety-like behavioral abnormality in aged TSOD mice. The results suggest that oleuropein suppresses the progression of type 2 diabetes and diabetes-related behavioral abnormality over the long-term.

Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3.

Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t1/2=9.8 to 2.8h), the half-life of C9-fused ELuc was significantly shorter (t1/2=1.0h) than that of PEST-fused ELuc when measurements were conducted at 37°C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.

Type 2 diabetes model TSOD mouse is exposed to oxidative stress at young age.

Tsumura Suzuki Obese Diabetes (TSOD) mouse, a model of obese type 2 diabetes, older than around 11 weeks of age develops diabetic phenotypes. Previous studies have indicated that the development of diabetes is partly due to three loci associated with body weight and glucose homeostasis. However, little is known about the initial events triggering the development of the diabetic phenotypes in TSOD mouse. Here, we investigated the alteration of diabetes-related parameters, including the levels of blood glucose and inflammatory cytokines, and the oxidative stress status, in young TSOD mice. TSOD mice at 5 weeks of age showed increases in body weight and plasma total cholesterol level, but not hyperglycemia or impaired glucose tolerance, compared with age-matched control Tsumura Suzuki Non-Obese (TSNO) mice. Plasma tumor necrosis factor (TNF)-α and interleukin (IL)-6 were not detected in TSOD mice at 5 weeks of age. However, plasma total hydroxyoctadecadienoic acid (tHODE), a biomarker of oxidative stress, was increased in TSOD mice relative to TSNO mice at same age. The results demonstrated that young TSOD mice are exposed to oxidative stress before developing the diabetic phenotypes, and suggested that oxidative stress is an initial event triggering the development of diabetes in TSOD mice.

ISL1 regulates peroxisome proliferator-activated receptor γ activation and early adipogenesis via bone morphogenetic protein 4-dependent and -independent mechanisms.

While adipogenesis is controlled by a cascade of transcription factors, the global gene expression profiles in the early phase of adipogenesis are not well defined. Using microarray analysis of gene expression in 3T3-L1 cells, we have identified evidence for the activity of 2,568 genes during the early phase of adipocyte differentiation. One of these, the ISL1 gene, was of interest since its expression was markedly upregulated 1 h after initiation of differentiation, with a subsequent rapid decline. Overexpression of ISL1 at early times during adipocyte differentiation but not at later times was found to profoundly inhibit differentiation. This was accompanied by moderate downregulation of peroxisome proliferator-activated receptor γ (PPARγ) levels, substantial downregulation of PPARγ downstream genes, and downregulation of bone morphogenetic protein 4 (BMP4) levels in preadipocytes. Readdition of BMP4 overcame the inhibitory effect of ISL1 on the expression of PPARγ but not aP2, a gene downstream of PPARγ, and BMP4 also partially rescued ISL1 inhibition of adipogenesis, an effect which is additive with rosiglitazone. These results suggest that ISL1 is intimately involved in early regulation of adipogenesis, modulating PPARγ expression and activity via BMP4-dependent and -independent mechanisms. Our time course gene expression survey sets the stage for further studies to explore other early and immediate regulators.

Dual-color bioluminescence imaging assay using green- and red-emitting beetle luciferases at subcellular resolution.

Bioluminescence imaging is widely used to monitor cellular events, including gene expression in vivo and in vitro. Moreover, recent advances in luciferase technology have made possible imaging at the single-cell level. To improve the bioluminescence imaging system, we have developed a dual-color imaging system in which the green-emitting luciferase from a Brazilian click beetle (Emerald Luc, ELuc) and the red-emitting luciferase from a railroad worm (Stable Luciferase Red, SLR) were used as reporters, which were localized to the peroxisome and the nucleus, respectively. We clearly captured simultaneously the subcellular localization of ELuc in the peroxisome and SLR in the nucleus of a single cell using a high-magnification objective lens with 3-min exposure time without binning using a combination of optical filters. Furthermore, to apply this system to quantitative time-lapse imaging, the activation of nuclear factor triggered by tumor necrosis factor α was measured using nuclear-targeted SLR and peroxisome-targeted ELuc as the test and internal control reporters, respectively. We successfully quantified the kinetics of activation of nuclear factor κB using nuclear-targeted SLR and the transcriptional change of the internal control promoter using peroxisome-targeted ELuc simultaneously in a single cell, and showed that the activation kinetics, including activation rate and amplitude, differed among cells. The results demonstrated that this imaging system can visualize the subcellular localization of reporters and track the expressions of two genes simultaneously at subcellular resolution.

Identification of the control region of pancreatic expression of Bmp4 in vitro and in vivo.

Bone morphogenetic protein 4 (Bmp4) was recently shown to be related to glucose homeostasis in mouse adult pancreas through the regulation of insulin production. We previously revealed the predominant expression of Bmp4 in adult pancreas by in vivo imaging of transgenic mice. However, the control regions for predominant Bmp4 expression in the adult pancreas are unclear. In this study, we established transgenic (Tg) mice that allow real time in vivo bioluminescence imaging of the enhancer/promoter activity of the Bmp4 gene. Tg mice expressing firefly luciferase with a 7 kb upstream region and 5'-non-coding sequence (three exons and two introns) of the Bmp4 gene showed pancreatic expression of bioluminescence, while the Tg mice bearing luciferase with the 7 kb upstream region alone did not show pancreatic expression of the reporter gene. Interestingly, pancreatic expression of bioluminescence was also present in Tg mice harboring the truncated promoter without exon IA and IB, indicating the presence of a cryptic promoter in front of exon II. Furthermore, the bioluminescence signal was not detected in embryonic pancreas, but increasing signals were observed in neonatal and infantile Tg mice depending on the genotypes observed. These results suggested that a novel mechanism of transcription is involved in pancreatic expression of the Bmp4 gene.

In vivo determination of vitamin d function using transgenic mice carrying a human osteocalcin luciferase reporter gene.

Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1 α ,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening.

A crucial role of bone morphogenetic protein signaling in the wound healing response in acute liver injury induced by carbon tetrachloride.

Background. Acute liver injury induced by administration of carbon tetrachloride (CCl(4)) has used a model of wound repair in the rat liver. Previously, we reported transient expression of bone morphogenetic protein (Bmp) 2 or Bmp4 at 6-24 h after CCl(4) treatment, suggesting a role of BMP signaling in the wound healing response in the injured liver. In the present study, we investigated the biological meaning of the transient Bmp expression in liver injury. Methods. Using conditional knockout mice carrying a floxed exon in the BMP receptor 1A gene, we determined the hepatic gene expressions and proliferative activity following CCl(4)-treated liver. Results. We observed retardation of the healing response in the knockout mice treated with CCl(4), including aggravated histological feature and reduced expressions of the albumin and Tdo2 genes, and a particular decrease in the proliferative activity shown by Ki-67 immunohistochemistry. Conclusion. Our findings suggest a crucial role of BMP signaling in the amelioration of acute liver injury.

Establishment and characterization of a transgenic mouse model for in vivo imaging of Bmp4 expression in the pancreas.

Type-2 diabetes results from the development of insulin resistance and a concomitant impairment of insulin secretion. Bone morphogenetic protein 4 (Bmp4)-Bmp receptor 1A signaling in ß cells has recently been reported to be required for insulin production and secretion. In addition, Bmp4 blocks the differentiation and promotes the expansion of endocrine progenitor cells. Bmp4 therefore regulates the maintenance of homeostasis in the pancreas. In this study, we constructed a reporter plasmid carrying 7-kb enhancer and promoter region of the Bmp4 gene upstream of the firefly luciferase gene. We used this construct to produce transgenic mice by pro-nuclear microinjection, for subsequent in vivo monitoring of Bmp4 expression. The bioluminescent signal was detected mainly in the pancreas in three independent lines of transgenic mice. Furthermore, the bioluminescent signal was enhanced in association with the autophagy response to 24-h fasting. These results suggest that pancreatic expression of Bmp4 is involved in responding to the physiological environment, including through autophagy. These mouse models represent useful tools for toxicological screening, and for investigating the mechanisms responsible for pancreatic Bmp4 functions in vivo, with relevance to improving our understanding of pancreatic diseases.

Establishment and characterization of hepatic stem-like cell lines from normal adult rat liver.

The liver is a unique organ with the potential to regenerate from injury. Hepatic stem cells contribute to liver regeneration when surviving hepatocytes in injured liver are unable to proliferate. To investigate the mechanism of liver regeneration in vitro, we established hepatic stem cell lines named HY1, HY2 and HY3, derived from a healthy liver of adult rat. HY cells showed an expression pattern similar to oval cells, and efficiently induced hepatic differentiation following sequential treatment with type I collagen, transforming growth factor-beta1 (TGF-beta1), and hepatocyte growth factor (HGF) or oncostatin M (OSM). These results suggested that HY cells are liver stem cells representing an excellent tool for in vitro studies on liver regeneration.