Masaki Nagaya

A Whole-Organ Regenerative Medicine Approach for Liver Replacement

BACKGROUND & AIMSnThe therapy of choice for end-stage liver disease is whole-organ liver transplantation, but this option is limited by a shortage of donor organs. Cell-based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation, but neither has proven effective to date. A regenerative medicine approach for liver replacement has recently been described that includes the use of a three-dimensional organ scaffold prepared by decellularization of xenogeneic liver. The present study investigates a new, minimally disruptive method for whole-organ liver decellularization and three different cell reseeding strategies to engineer functional liver tissue.nnnMETHODSnA combination of enzymatic, detergent, and mechanical methods are used to remove all cells from isolated rat livers. Whole-organ perfusion is used in a customized organ chamber and the decellularized livers are examined by morphologic, biochemical, and immunolabeling techniques for preservation of the native matrix architecture and composition. Three different methods for hepatocyte seeding of the resultant three-dimensional liver scaffolds are evaluated to maximize cell survival and function: (1) direct parenchymal injection, (2) multistep infusion, or (3) continuous perfusion.nnnRESULTSnThe decellularization process preserves the three-dimensional macrostructure, the ultrastructure, the composition of the extracellular matrix components, the native microvascular network of the liver, and the bile drainage system, and up to 50% of growth factor content. The three-dimensional liver matrix reseeded with the multistep infusion of hepatocytes generated ∼90% of cell engraftment and supported liver-specific functional capacities of the engrafted cells, including albumin production, urea metabolism, and cytochrome P450 induction.nnnCONCLUSIONSnWhole-organ liver decellularization is possible with maintenance of structure and composition suitable to support functional hepatocytes.

Barriers to the successful treatment of liver disease by hepatocyte transplantation

Management of patients with hepatic failure and liver-based metabolic disorders is complex and expensive. Hepatic failure results in impaired coagulation, altered consciousness and cerebral function, a heightened risk of multiple organ system failure, and sepsis [1]. Such manifold problems are only treatable today and for the foreseeable future by transplantation. In fact, whole or auxiliary partial liver transplantation is often the only available treatment option for severe, even if transient, hepatic failure. Patients with life-threatening liver-based metabolic disorders similarly require organ transplantation even though their metabolic diseases are typically the result of a single enzyme deficiency, and the liver otherwise functions normally. For all of the benefits it may confer, liver transplantation is not an ideal therapy, even for severe hepatic failure. More than 17,000 patients currently await liver transplantation in the United States, a number that seriously underestimates the number of patients that need treatment [2], as it has been estimated that more than a million patients could benefit from transplantation [3]. Unfortunately, use of whole liver transplantation to treat these disorders is limited by a severe shortage of donors and by the risks to the recipient associated with major surgery [4].

Dendritic cell-based immunotherapy targeting synthesized peptides for advanced biliary tract cancer.

BackgroundThe aim of this retrospective study was to clarify the safety and efficacy of dendritic cell (DC)-based immunotherapy targeting synthesized peptides, Wilms tumor 1 (WT1) and Mucin 1, cell surface associated (MUC1) for biliary tract cancers (BTCs).MethodsSixty-five patients who had nonresectable, recurrent, or metastatic BTCs and received the DC-based immunotherapy were selected for the study. DCs were pulsed with WT1 and/or MUC1. The adverse events (AEs) and clinical responses were examined.ResultsNo serious treatment-related AEs were observed. Median survival time (MST) from diagnosis and from the first vaccination was 18.5 and 7.2xa0months, respectively. By multivariate Cox proportional hazard analysis, the significant independent factors were found to be (1) combined chemotherapy, (2) albumin level ≥4.0 g/dL before vaccination, (3) C-reactive protein level <0.5xa0mg/dL before vaccination, and (4) fever after vaccination. The MST from the first vaccination with or without chemotherapy was 8.2 and 5.3xa0months, respectively (Pu2009=u20090.016), and MST for the patients with prognostic nutritional index ≥40 and <40 was 8.1 and 5.0xa0months, respectively (Pu2009=u20090.023).ConclusionsAlthough a small uncontrolled nonrandomized study, DC-based immunotherapy for BTCs was safe and produced a clinical response for the patients who underwent chemotherapy and maintained a good nutrition status.

The microenvironment in hepatocyte regeneration and function in rats with advanced cirrhosis.

In advanced cirrhosis, impaired function is caused by intrinsic damage to the native liver cells and from the abnormal microenvironment in which the cells reside. The extent to which each plays a role in liver failure and regeneration is unknown. To examine this issue, hepatocytes from cirrhotic and age‐matched control rats were isolated, characterized, and transplanted into the livers of noncirrhotic hosts whose livers permit extensive repopulation with donor cells. Primary hepatocytes derived from livers with advanced cirrhosis and compensated function maintained metabolic activity and the ability to secrete liver‐specific proteins, whereas hepatocytes derived from cirrhotic livers with decompensated function failed to maintain metabolic or secretory activity. Telomere studies and transcriptomic analysis of hepatocytes recovered from progressively worsening cirrhotic livers suggest that hepatocytes from irreversibly failing livers show signs of replicative senescence and express genes that simultaneously drive both proliferation and apoptosis, with a later effect on metabolism, all under the control of a central cluster of regulatory genes, including nuclear factor κB and hepatocyte nuclear factor 4α. Cells from cirrhotic and control livers engrafted equally well, but those from animals with cirrhosis and failing livers showed little initial evidence of proliferative capacity or function. Both, however, recovered more than 2 months after transplantation, indicating that either mature hepatocytes or a subpopulation of adult stem cells are capable of full recovery in severe cirrhosis. Conclusion: Transplantation studies indicate that the state of the host microenvironment is critical to the regenerative potential of hepatocytes, and that a change in the extracellular matrix can lead to regeneration and restoration of function by cells derived from livers with end‐stage organ failure. (HEPATOLOGY 2011)

Impact of dendritic cell vaccines pulsed with Wilms' tumour-1 peptide antigen on the survival of patients with advanced non-small cell lung cancers

PURPOSEnDendritic cell (DC)-based vaccines have been expected to serve as new therapeutic approaches for advanced non-small cell lung cancers (NSCLCs); however, their clinical outcomes have not been fully elucidated. We report a single-centre clinical study analysing factors affecting the survival of patients with advanced NSCLCs who received DC vaccines pulsed with or without Wilms tumour protein-1 (WT1) peptide.nnnMETHODSnAmong 62 patients with previously treated inoperable or postoperatively relapsed NSCLCs who met the inclusion criteria, DCs from 47 (76%) patients who showed HLA-A2402/0201/0206 were pulsed with one or more corresponding WT1 peptide antigens. DC vaccines were intradermally injected biweekly.nnnRESULTSnClinical responses based on response evaluation criteria in solid tumours (RECIST) were found in 31 (50%) patients at 3 months after the first DC vaccine (complete response: 1 (1.6%), partial response: 4 (6.5%), stable disease: 26 (41.9%)). Median survival time was 27 months (82% in 1 year and 54% in 2 years) from initial diagnosis, and that was 12 months (48% in 1 year and 22% in 2 years) from the first DC vaccination. Importantly, multivariate analyses revealed that only two factors, blood haemoglobin and the use of WT1 peptides, significantly affected the overall survival of patients from both initial diagnosis and first vaccination.nnnCONCLUSIONSnThis study is the first to suggest that DC vaccines pulsed with WT1 may hold a significant impact to prolong the overall survival of patients with advanced NSCLCs.

Induced pluripotent stem cells model personalized variations in liver disease resulting from α1‐antitrypsin deficiency

In the classical form of α1‐antitrypsin deficiency (ATD), aberrant intracellular accumulation of misfolded mutant α1‐antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain‐of‐function, “proteotoxic” mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation, others with the same genetic defect completely escape this clinical phenotype. We investigated whether induced pluripotent stem cells (iPSCs) from ATD individuals with or without SLD could model these personalized variations in hepatic disease phenotypes. Patient‐specific iPSCs were generated from ATD patients and a control and differentiated into hepatocyte‐like cells (iHeps) having many characteristics of hepatocytes. Pulse‐chase and endoglycosidase H analysis demonstrate that the iHeps recapitulate the abnormal accumulation and processing of the ATZ molecule, compared to the wild‐type AT molecule. Measurements of the fate of intracellular ATZ show a marked delay in the rate of ATZ degradation in iHeps from SLD patients, compared to those from no liver disease patients. Transmission electron microscopy showed dilated rough endoplasmic reticulum in iHeps from all individuals with ATD, not in controls, but globular inclusions that are partially covered with ribosomes were observed only in iHeps from individuals with SLD. Conclusion: iHeps model the individual disease phenotypes of ATD patients with more rapid degradation of misfolded ATZ and lack of globular inclusions in cells from patients who have escaped liver disease. The results support the concept that “proteostasis” mechanisms, such as intracellular degradation pathways, play a role in observed variations in clinical phenotype and show that iPSCs can potentially be used to facilitate predictions of disease susceptibility for more precise and timely application of therapeutic strategies. (Hepatology 2015;62:147‐157)

Prognostic factors related to add-on dendritic cell vaccines on patients with inoperable pancreatic cancer receiving chemotherapy: A multicenter analysis

AbstractObjectivenDendritic cell (DC)-based cancer vaccines may have a significant benefit to patients with advanced pancreatic cancer. However, variations among clinical studies make it difficult to compare clinical outcomes. Here, we identified factors that determined the clinical benefits by analyzing data obtained at seven Japanese institutions that employed the same DC preparation and treatment regimens.MethodsOf 354 patients who met the inclusion criteria, 255 patients who received standard chemotherapy combined with peptide-pulsed DC vaccines were analyzed.ResultsThe mean survival time from diagnosis was 16.5xa0months (95xa0% CI 14.4–18.5) and that from the first vaccination was 9.9xa0months (95xa0% CI 8.0–12.9). Known prognostic baseline factors related to advanced pancreatic cancer, namely ECOG-PS, peritoneal metastasis, liver metastasis, and the prognostic nutrition index, were also representative. Importantly, we found that erythema reaction after vaccination was an independent and treatment-related prognostic factor for better survival and that OK-432 might be a good adjuvant enhancing the antitumor immunity during DC vaccination.ConclusionsThis is the first report of a multicenter clinical study suggesting the feasibility and possible clinical benefit of an add-on DC vaccine in patients with advanced pancreatic cancer who are undergoing chemotherapy. These findings need to be addressed in well-controlled prospective randomized trials.

The feasibility and clinical effects of dendritic cell-based immunotherapy targeting synthesized peptides for recurrent ovarian cancer

BackgroundDespite the increased rate of complete response to initial chemotherapy, most patients with advanced ovarian cancer relapse and succumb to progressive disease. Dendritic cell (DC)-based immunotherapy has been developed as a novel strategy for generating antitumor immunity as part of cancer treatments. The present study aimed to assess the feasibility and clinical effects of DC therapy for recurrent ovarian cancer (ROC).MethodsThis retrospective study included 56 ROC patients who initially received standard chemotherapy followed by DC-based immunotherapy targeting synthesized peptides at 2 institutions between March 2007 and August 2013. The adverse events (AEs) and clinical responses were examined.ResultsNo serious treatment-related AEs were observed. Seventy one percent of the enrolled patients developed an immunologic response. The median survival time (MST) from ROC diagnosis was 30.4xa0months, and that from the first vaccination was 14.5xa0months. Albumin levels of ≥4.0xa0g/dL and lactate dehydrogenase levels of <200xa0IU/L before vaccination were identified as significant independent factors by multivariate Cox proportional hazard analysis. The MST from the first vaccination in patients with albumin levels of ≥4.0 and <4.0xa0g/dL were 19.9 and 11.6xa0months, respectively. The corresponding disease control rates were 36% and 15%, respectively.ConclusionsOur results demonstrated the feasibility and potential clinical effectiveness of DC-based immunotherapy for ROC patients. Additionally, a good nutritional status might be an important factor for further clinical effects.

Evaluation of Thermoreversible Gelation Polymer for Regeneration of Focal Liver Injury

Liver injuries are often associated with complications including infection of the dead space, bleeding, leakage of bile and so on. We have recently developed a thermoreversible gelation polymer (TGP) which provides a good healing environment for wounds and possibly reduces complications. The purpose of this study was to evaluate whether adequate regeneration occurred with a liver defect by using TGP. The sol-gel transition of TGP is reversibly controlled by temperature; TGP is soluble below a lower critical solution temperature (LCST) of 22°C, and becomes solid above the LCST. Soluble TGP can reach anywhere, and gelation of TGP occurs at the wound surface by body temperature to fill the wound/cavity. A section of median part of the left lobe comprising 3% (2 × 2 cm wide and 1 cm deep) of the liver was resected, and the Beagle dogs were assigned to three groups: ‘resection alone group’, ‘resection + fibrin glue (FG) group’ and ‘resection + TGP group’. The resection alone group and the resection + FG group showed severe fibrosis at week 12, and a scarring was clearly visible. The resection + TGP group showed almost complete healing by week 4, with no adhesion and recession of the wound; the resection site was completely filled with TGP, liver-like capsule organoids emerged to cover the wound and neovascularization was observed within the organoids. Furthermore, the resected liver regenerated completely by week 12, TGP was replaced by hepatocytes, and the presence of hepatic lobules confirmed structural reorganization. The number of RCA-1-positive macrophages accumulating around the wound was significantly reduced in the resection + TGP group compared to the other two groups. In the early stage of liver resection and regeneration, TGP seemed to suppress the accumulation of macrophages and stellate cells. In the late stage, when massive inflammatory cell accumulation had subsided, TGP was degraded, that may contribute to avoid unnecessary inhibition of the liver regeneration process. Collectively, TGP may induce efficient regeneration by reducing the fibrosis and enhancing proliferation, even with a minor liver defect. Because TGP has good biocompatibility, it may become useful as an ideal biomaterial for the treatment of liver injuries.

Thermoreversible gelation polymer induces the emergence of hepatic stem cells in the partially injured rat liver.

Focal injury of the adult liver causes formation of granulomatous tissue and fibrosis. When thermoreversible gelation polymer (TGP) was applied to such defects of the rat liver, complete recovery of hepatic tissues was observed without granulation. We analyzed the mechanism of the regeneration. TGP is a chemically synthesized biocompatible polymer material whose sol‐gel transition is reversible by changing the temperature. Cooled TGP was poured into a penetration lesion of the rat liver. Immunohistochemistry and polymerase chain reaction were carried out using tissues and cultured cells isolated from ductular structures. Immunocytochemical and ultrastructural analyses were also conducted. Seven days after TGP treatment, ductular reactions were observed around the wound and ductules elongated to the injured area. Cells in the structures were alpha‐fetoprotein (AFP) positive, albumin+, CK19+, c‐Kit+, and Thy1+. Hepatocyte‐like cells possessing glycogen appeared around the tips of the ductules from day 9. The defect was completely replaced with hepatocytes by day 28. Cells isolated from the ductules expressed Musashi‐1, c‐Kit, Thy1, AFP, albumin, transferrin, connexin 43, and CK19. When the cultured cells were covered by TGP, they rapidly proliferated to form colonies, whereas without TGP cells gradually died. Morphologically and ultrastructurally the cells were similar to hepatocytes. They expressed not only albumin and transferrin but TAT, CYP2E1, and CCAAT/enhancer binding protein α. Some cells formed bile canaliculus‐like structures. In conclusion, TGP may trigger the initiation of hepatic stem cells in biliary ductules, and stem cell activation may occur even in the regeneration of the normal liver. (HEPATOLOGY 2006;43:1053–1062.)