Improvement in aged liver regeneration using cell transplantation with chemically induced liver progenitors.

BACKGROUND: A decrease in the regenerative capacity of age-damaged liver tissue has been reported. Liver progenitor cells may play an important role in the regeneration of injured livers. In the present study we aimed to investigate improvements in the regenerative capacity of age-damaged livers using chemically induced liver progenitors (CLiPs) derived from mature hepatocytes. METHODS: Old (>90 weeks) and young (<20 weeks) mice underwent 70% hepatectomy, with or without trans-splenic CLiP administration. The residual liver/bodyweight (LW/BW) ratio was measured on postoperative days 1 and 7, and changes in liver regeneration and histology were evaluated. RESULTS: At 7 days post-hepatectomy, LW/BW ratios were significantly better in CLiP-treated old mice than in untreated old mice (p = .02). By contrast, no effect of CLiP transplantation was observed in young mice (p = .62). Immunofluorescence staining of liver tissue after CLiP administration showed an increase in Ki67-positive cells (p < .01). Flow cytometry analysis of green fluorescent protein-labeled CLiPs indicated that transplanted CLiPs differentiated into mature hepatocytes and were present in the recipient liver. CONCLUSIONS: CLiP transplantation appears to ameliorate the age-related decline in liver regeneration in mice.

Three-dimensional human bile duct formation from chemically induced human liver progenitor cells.

Background: The intrahepatic bile ducts (BDs) play an important role in the modification and transport of bile, and the integration between the BD and hepatocytes is the basis of the liver function. However, the lack of a source of cholangiocytes limits in vitro research. The aim of the present study was to establish three-dimensional BDs combined with human mature hepatocytes (hMHs) in vitro using chemically induced human liver progenitor cells (hCLiPs) derived from hMHs. Methods: In this study, we formed functional BDs from hCLiPs using hepatocyte growth factor and extracellular matrix. BDs expressed the typical biliary markers CK-7, GGT1, CFTR and EpCAM and were able to transport the bile-like substance rhodamine 123 into the lumen. The established three-dimensional BDs were cocultured with hMHs. These cells were able to bind to the BDs, and the bile acid analog CLF was transported from the culture medium through the hMHs and accumulated in the lumen of the BDs. The BDs generated from the hCLiPs showed a BD function and a physiological system (e.g., the transport of bile within the liver) when they were connected to the hMHs. Conclusion: We present a novel in vitro three-dimensional BD combined with hMHs for study, drug screening and the therapeutic modulation of the cholangiocyte function.

Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells.

Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4+CD25+ Tregs and CD4+Foxp3+ Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.

Therapeutic effect and mechanism of Daikenchuto in a model of methotrexate-induced acute small intestinal mucositis.

BACKGROUND: Daikenchuto (DKT) has positive therapeutic effects on improving various gastrointestinal disorders. The present study investigated whether or not DKT has a potential therapeutic effect on chemotherapy-induced acute small intestinal mucositis (CIM) in a rat model. METHODS: Intraperitoneal injection of 10 mg/kg methotrexate (MTX) every 3 days for a total of 3 doses was used for induction of CIM in a rat model. The MTX and DKT-MTX groups were injected with MTX as above from the first day, and the DKT-MTX and DKT groups were administered 2.7% DKT via the diet at the same time. The rats were euthanized on day 15. RESULTS: The DKT-MTX group showed an improvement in the body weight and conditions of gastrointestinal disorders as well as increased levels of diamine oxidase in plasma and in the small intestinal villi. The pathology results showed that small intestinal mucosal injury in the DKT-MTX group was less severe than that in the MTX group. Immunohistochemistry for myeloperoxidase and malondialdehyde and quantitative real-time polymerase chain reaction (RT-qPCR) for TGF-ß1 and HIF-1α showed that DKT attenuated peroxidative damage. The crypts in the DKT-MTX group contained more Ki-67-positive cells than MTX group. The zonula occluden-1 and claudin-3 results showed that DKT promoted repair of the mucosal barrier. RT-qPCR for the amino acid transporters EAAT3 and BO+AT also confirmed that DKT promoted mucosal repair and thus promoted nutrient absorption. CONCLUSION: DKT protected against MTX-induced CIM in a rat model by reducing inflammation, stimulating cell proliferation, and stabilizing the mucosal barrier.

Possible Use of Non-purine Selective Xanthine Oxidoreductase Inhibitors for Prevention of Exercise-induced Acute Kidney Injury Associated with Renal Hypouricemia.

Exercise-induced acute kidney injury (EIAKI) is frequently complicated with renal hypouricemia (RHUC). In patients with RHUC, limiting anaerobic exercise can prevent EIAKI. However, it is challenging to reduce exercise intensity in athletes. We herein report a 16-year-old Japanese football player with familial RHUC with compound heterozygous mutations in urate transporter 1 (URAT1) who presented with recurrent EIAKI. As prophylaxis (hydration during exercise) could not prevent EIAKI, febuxostat was initiated. EIAKI was not observed for 16 months despite exercising intensively. Hence, non-purine-selective xanthine oxidoreductase inhibitors may decrease the incidence of EIAKI in athletes with RHUC.

Outcomes of pediatric obesity-related lifestyle disease screenings in Kumamoto.

BACKGROUND: Obesity is an important issue causing both health hazards and socioeconomic loss to those affected. Kumamoto City regularly performs obesity-related lifestyle disease screenings for fourth grade children with obesity, including physical examinations, blood tests, and special examination referrals. We retrospectively analyzed the outcomes of the screenings conducted from 2011 to 2020. METHODS: The percentage of overweight was calculated using data from the Lifestyle Disease Screening Board of Kumamoto City from 2011 to 2020. The percentage of overweight, abdominal circumference, blood pressure, and laboratory test outcomes of the Secondary Lifestyle Disease Test were evaluated. RESULTS: The proportion of children with obesity in grades 1-4 in Kumamoto was higher than the national average, while that in grades 5-6 was lower than the national average. Among the fourth graders screened, 6521 were eligible for the Secondary Lifestyle Disease Tests, of which 3291 children underwent the test. In the testing, 22.3% of the boys and 29.1% of the girls were nonobese. Moreover, 25.9% of the boys and 19.2% of the girls, including nonobese children, required further examination and intervention. Notably, 62.1% of the boys and 46.2% of the girls who were nonobese and required special examination had a waist circumference of ≥75 cm or waist-to-height ratios of ≥0.5. CONCLUSIONS: Obesity-related lifestyle disease screenings contributed to preventing obesity progression. Abdominal circumference measurements may be useful in determining nonobese children at a risk of lifestyle diseases.

Stress exacerbates pancreatic cancer both directly and indirectly by creating an immunosuppressive environment.

BACKGROUND/PURPOSE: Sympathetic nerve stimulation by stress exacerbates various solid tumors, including pancreatic cancer (PCa). The relationship between cancer and immunity has been suggested; however, there is limited information about the effects of nerve stimulation on immunity and cancer. We aimed to investigate the involvement of sympathetic nerve stimulation in immune cells and its effects on PCa using a restraint stress mouse model. METHODS: In the in vitro experiment, the mouse-derived PCa cell line (LTPA) was cultured in a noradrenalin-supplemented medium. In the in vivo experiment, mice were divided into non-stress and stress groups. RESULTS: LTPA proliferated significantly more when cultured in a noradrenalin-supplemented medium than in a normal medium. Flow cytometry analysis of blood immune cells revealed a significant decrease in B cells, T cells, and macrophages and a significant increase in myeloid-derived suppressor cells (MDSCs) in the stress group. Furthermore, a significant increase in blood noradrenaline levels was observed in the stress group (p < .01). In the PCa mice model, immune cells in the blood showed a similar trend, and the stress group had a poor prognosis. Furthermore, immunostaining at the tumor site showed that there was a lower number of B and T cells in the stress group. In addition, MDSCs were present at the tumor margins. CONCLUSION: These results suggest that sympathetic nerve stimulation is not only directly involved in PCa growth but also exacerbates PCa by creating an immunosuppressive environment in the blood and tumor tissue.

Neural circuit plasticity for complex non-declarative sensorimotor memory consolidation during sleep.

Evidence is accumulating that the brain actively consolidates long-term memory during sleep. Motor skill memory is a form of non-declarative procedural memory and can be coordinated with multi-sensory processing such as visual, tactile, and, auditory. Conversely, perception is affected by body movement signal from motor brain regions. Although both cortical and subcortical brain regions are involved in memory consolidation, cerebral cortex activity can be recorded and manipulated noninvasively or minimally invasively in humans and animals. NREM sleep, which is important for non-declarative memory consolidation, is characterized by slow and spindle waves representing thalamo-cortical population activity. In animals, electrophysiological recording, optical imaging, and manipulation approaches have revealed multi-scale cortical dynamics across learning and sleep. In the sleeping cortex, neural activity is affected by prior learning and neural circuits are continually reorganized. Here I outline how sensorimotor coordination is formed through awake learning and subsequent sleep.

Transplantation of chemically-induced liver progenitor cells ameliorates hepatic fibrosis in mice with diet-induced nonalcoholic steatohepatitis.

Chemically-induced liver progenitors (CLiPs) have promising applications in liver regenerative medicine. We aimed to clarify the efficacy of CLiPs for ameliorating fibrosis in a diet-induced nonalcoholic steatohepatitis rat model, since nonalcoholic fatty liver disease is currently recognized as the most common form of chronic liver disease in developed countries. METHODS: Primary mature hepatocytes were isolated from 7-week-old male Wistar rats. To establish CLiPs, isolated hepatocytes were cultured in differentiation medium composed of Y-27632, A-83-01, and CHIR99021 (YAC medium). As an animal model that reproduces NASH pathophysiology, 6-week-old severe combined immunodeficient (SCID) mice were carefully selected and prepared and fed with choline-deficient, L-amino acid-defined, high-fat diet (HFD). After 12 weeks' HFD feeding, the mice were assigned to continue HFD with or without the administration of rat CLiPs (HFD + CLiPs and HFD-CLiPs, respectively). Rat CLiPs were administered from the spleen. Hepatic fibrosis was semi-quantitatively evaluated according to histology. Liver parenchyma and blood samples were collected for biochemical analyses. RESULTS: Rat CLiPs were positive for CK19 and EpCAM were successfully delivered to the liver. At 8 weeks after CLiPs transplantation, the HFD + CLiPs group showed significantly less positive staining than the HFD-CLiPs group. Alanine aminotransferase significantly improved in the HFD + CLiPs group, as demonstrated by Azan staining and αSMA immunostaining. RT qPCR showed that the liver expression of MMP2 and 9 tended to be higher in the HFD + CLiPs group. CONCLUSIONS: The anti-fibrotic effect of CLiPs was demonstrated in the immunodeficient NASH animal model and may have therapeutic applications in humans.

Airway management in the pediatric emergency department in Japan: A multicenter prospective observational study.

Aim: Tracheal intubation is a vital resuscitation procedure in the pediatric emergency department (ED). Despite its importance, little is known about the current status of emergency airway management in Japan. In this context, we aimed to investigate the airway management characteristics-particularly the location, patient, and provider factors-in the pediatric ED. Methods: We conducted a multicenter, prospective study of five pediatric EDs in Japan from October 2018 to June 2020. The study included all children (aged ≤18 years) who underwent intubation at the pre-ED or ED setting by physicians and those who were transferred from the ED to the operation room (OR) or pediatric intensive care unit (PICU) for intubation. We described the airway management characteristics according to the location, patient, and provider factors. Results: Of 231 children, 9 (4%) were transferred to the OR or PICU for airway management. Among the remaining 222 children, 45 were intubated at the pre-ED setting and 177 were intubated in the ED. The overall first-attempt success rate was 72%, with the rate varying by location, patient, and provider factors-for example, 68% at the pre-ED setting, 67% for children <2 years, 56% for children with airway-related anatomical anomalies, and 61% with intubation by a resident physician. Intubation-related adverse events were observed in 17%, most of which were hypoxemia (14%). Conclusions: Based on data from a multicenter prospective study, the overall first-attempt intubation success rate in pediatric EDs in Japan was 72%, with large variations by location, patient, and provider factors.

Analysis of Purine Metabolism to Elucidate the Pathogenesis of Acute Kidney Injury in Renal Hypouricemia.

Renal hypouricemia is a disease caused by the dysfunction of renal urate transporters. This disease is known to cause exercise-induced acute kidney injury, but its mechanism has not yet been established. To analyze the mechanism by which hypouricemia causes renal failure, we conducted a semi-ischemic forearm exercise stress test to mimic exercise conditions in five healthy subjects, six patients with renal hypouricemia, and one patient with xanthinuria and analyzed the changes in purine metabolites. The results showed that the subjects with renal hypouricemia had significantly lower blood hypoxanthine levels and increased urinary hypoxanthine excretion after exercise than healthy subjects. Oxidative stress markers did not differ between healthy subjects and hypouricemic subjects before and after exercise, and no effect of uric acid as a radical scavenger was observed. As hypoxanthine is a precursor for adenosine triphosphate (ATP) production via the salvage pathway, loss of hypoxanthine after exercise in patients with renal hypouricemia may cause ATP loss in the renal tubules and consequent tissue damage.

Optical imaging and manipulation of sleeping-brain dynamics in memory processing.

During sleep, the brain undergoes transitions of activity states and reorganization of neural circuits. Recent in vivo optical imaging and manipulation techniques are revealing spatiotemporal and multiscale brain dynamics during sleep and their functional roles in awake state cognition, including learning and memory. First, along with electrophysiological recordings, Ca2+ imaging is becoming the gold standard for tracking the activity of large neural ensembles to analyze memory replay during the sleep/wake cycle in freely moving and head-fixed animals. Comparable to the speed of electrophysiological recordings, voltage indicators can monitor neural activity at millisecond resolution. While one-photon systems have advantages in temporal resolution, two-photon lasers can image microstructures such as dendritic spines and axonal terminals in vivo. Also, optogenetic manipulation is used to perform loss- or gain-of-function experiments with temporal precision, cell-type, and pathway specificity without the artifacts observed with electrophysiological stimulation. In particular, closed-loop optogenetic manipulation is well suited to reveal the functional roles of neural activity and synaptic plasticity in each sleep/wake state. Here, I describe the recent advances in optical imaging and manipulation approaches for sleeping-brain dynamics in memory processing.

Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A.

Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain-derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl-) cells through genome editing and compared HCV production between Abl- cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl-NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.

Successful induction of human chemically induced liver progenitors with small molecules from damaged liver.

BACKGROUND: Human chemically induced liver progenitors (hCLiP) induced by small molecules produced by mature hepatocytes can potentially overcome issues related to hepatocyte transplantation, such as graft rejection or donor shortage. However, to our knowledge, no studies have explored the induction of hCLiP from mature hepatocytes (MHs) in damaged liver, indicated for liver transplantation. METHODS: Liver tissues were collected from surgically resected livers, including damaged livers, of 86 patients at our department, and hepatocytes were isolated using the collagenase perfusion method. Hepatocytes isolated from 33 of these 86 donors were cultured in YAC medium containing Y-27632 (ROCK inhibitor), A-83-01 (TGF-ß type I receptor inhibitor), and CHIR99021 (GSK-3 inhibitor) to induce hCLiP, and their functions were assessed. RESULTS: Hepatocytes were isolated regardless of the liver fibrosis classifications (viability: F0,1: 87.2 ± 13.2%; F2,3: 87.8 ± 13.1%; and F4: 86.3 ± 4.2%). Most hepatocytes cultured in the YAC medium acquired the liver progenitor cell (LPC) gene. The expression of MH markers (ALB, HNF4α, G6PC, and CYP1A2) was lower in hCLiP than in MHs before reprogramming. Reverse transcription-polymerase chain reaction revealed that hCLiP markers (e.g., EpCAM, SOX9, CK19, and CD133) exhibited higher expression in LPCs than in MHs. Furthermore, hCLiPs had the ability to differentiate into hepatocytes, and were engrafted on the liver surface as mature hepatocytes. CONCLUSION: Hepatocytes could be isolated from damaged liver. Furthermore, hCLiP may be obtained from hepatocytes isolated from damaged liver and may differentiate into MHs in vitro. Autologous hCLiP can potentially be transplanted without tumorigenesis and remodel damaged liver.

Effect of Pore Size of Honeycomb-Patterned Polymer Film on Spontaneous Formation of 2D Micronetworks by Coculture of Human Umbilical Vein Endothelial Cells and Mesenchymal Stem Cells.

The geometrical control of micronetwork structures ( µ NSs) formed by endothelial cells is an important topic in tissue engineering, cell-based assays, and fundamental biological studies. In this study, µ NSs are formed using human umbilical vein endothelial cells (HUVECs) by the coculture of HUVECs and human mesenchymal stem cells (MSCs) confined in a honeycomb-patterned poly-l-lactic acid film (honeycomb film (HCF)), which is a novel cell culture scaffold. The HCF is produced using the breath figure method, which uses condensed water droplets as pore templates. The confinement of the HUVECs and MSCs in the HCF along with the application of centrifugal force results in µ NS formation when the pore size is more than 20  µ m. Furthermore, µ NS development is geometrically restricted by the hexagonally packed and connected pores in the horizontal direction of the HCF. Network density is also controlled by changing the seeding density of the HUVECs and MSCs. The threshold pore size indicates that µ NSs can be formed spontaneously by using an HCF with a perfectly uniform porous structure. This result provides an important design guideline for the structure of porous cell culture scaffolds by applying a blood vessel model in vitro.

Net decrease in spine-surface GluA1-containing AMPA receptors after post-learning sleep in the adult mouse cortex.

The mechanisms by which sleep benefits learning and memory remain unclear. Sleep may further strengthen the synapses potentiated by learning or promote broad synaptic weakening while protecting the newly potentiated synapses. We tested these ideas by combining a motor task whose consolidation is sleep-dependent, a marker of synaptic AMPA receptor plasticity, and repeated two-photon imaging to track hundreds of spines in vivo with single spine resolution. In mouse motor cortex, sleep leads to an overall net decrease in spine-surface GluA1-containing AMPA receptors, both before and after learning. Molecular changes in single spines during post-learning sleep are correlated with changes in performance after sleep. The spines in which learning leads to the largest increase in GluA1 expression have a relative advantage after post-learning sleep compared to sleep deprivation, because sleep weakens all remaining spines. These results are obtained in adult mice, showing that sleep-dependent synaptic down-selection also benefits the mature brain.

Functional changes of cocultured hepatocyte sheets subjected to continuous liver regeneration stimulation in cDNA-uPA/SCID mouse: Differences in transplantation sites.

AIM: The formation of a secondary liver is expected in ectopic transplants in liver therapy. It is reported that the transplantation of hepatocyte sheets constitutes one of the techniques used to form a secondary liver. Accordingly, we established a subcutaneous transplant for hepatocyte/fibroblast sheets in previous studies. In this development study with hepatocyte/fibroblast sheets, we evaluated the differences in transplantation sites to promote the maturation of transplanted tissue in a liver injury model. METHODS: A cocultured hepatocyte sheet of fibroblasts (TIG-118 cells) and human hepatocytes (PXB cells) was prepared on a temperature-responsive culture dish. The prepared cocultured hepatocyte sheet was either transplanted subcutaneously or on the liver surface of a persistent liver injury model (cDNA-uPA/SCID mouse: uPA mouse), and was evaluated by the human albumin concentration in mouse blood. As a control group, hepatocyte cell sheets were used that were transplanted to both areas and compared. RESULTS: Although the cocultured hepatocyte sheet led to functional improvements in the early stages of culture in subcutaneous transplantation, these did not last in the long-term after transplantation. Although coculture effects were not observed in the liver surface transplantation case, long-term functional expressions in mono- and cocultured sheets in the case of liver surface transplantation were exhibited compared with subcutaneous administration. CONCLUSION: These results suggest that sustained stimulation of liver regenerationvaries depending on the transplant site and is largely involved in the maturation of hepatocyte tissue.

Bioengineering of a CLiP-derived tubular biliary-duct-like structure for bile transport in vitro.

The integration of a bile drainage structure into engineered liver tissues is an important issue in the advancement of liver regenerative medicine. Primary biliary cells, which play a vital role in bile metabolite accumulation, are challenging to obtain in vitro because of their low density in the liver. In contrast, large amounts of purified hepatocytes can be easily acquired from rodents. The in vitro chemically induced liver progenitors (CLiPs) from primary mature hepatocytes offer a platform to produce biliary cells abundantly. Here, we generated a functional CLiP-derived tubular bile duct-like structure using the chemical conversion technology. We obtained an integrated tubule-hepatocyte tissue via the direct coculture of hepatocytes on the established tubular biliary-duct-like structure. This integrated tubule-hepatocyte tissue was able to transport the bile, as quantified by the cholyl-lysyl-fluorescein assay, which was not observed in the un-cocultured structure or in the biliary cell monolayer. Furthermore, this in vitro integrated tubule-hepatocyte tissue exhibited an upregulation of hepatic marker genes. Together, these findings demonstrated the efficiency of the CLiP-derived tubular biliary-duct-like structures regarding the accumulation and transport of bile.

Regenerative medicine for the hepatobiliary system: A review.

Liver transplantation, the only proven treatment for end-stage liver disease and acute liver failure, is hampered by the scarcity of donors. Regenerative medicine provides an alternative therapeutic approach. Tremendous efforts dedicated to liver regenerative medicine include the delivery of transplantable cells, microtissues, and bioengineered whole livers via tissue engineering and the maintenance of partial liver function via extracorporeal support. This brief review summarizes the current status of regenerative medicine for the hepatobiliary system. For liver regenerative medicine, the focus is on strategies for expansion of transplantable hepatocytes, generation of hepatocyte-like cells, and therapeutic potential of engineered tissues in liver disease models. For biliary regenerative medicine, the discussion concentrates on the methods for generation of cholangiocyte-like cells and strategies in the treatment of biliary disease. Significant advances have been made in large-scale and long-term expansion of liver cells. The development of tissue engineering and stem cell induction technology holds great promise for the future treatment of hepatobiliary diseases. The application of regenerative medicine in liver still lacks extensive animal experiments. Therefore, a large number of preclinical studies are necessary to provide sufficient evidence for their therapeutic effectiveness. Much remains to be done for the treatment of hepatobiliary diseases with regenerative medicine.