Osamu Itano

Human-Scale Whole-Organ Bioengineering for Liver Transplantation: A Regenerative Medicine Approach

At this time, the only definitive treatment of hepatic failure is liver transplantation. However, transplantation has been limited by the severely limited supply of human donor livers. Alternatively, a regenerative medicine approach has been recently proposed in rodents that describe the production of three-dimensional whole-organ scaffolds for assembly of engineered complete organs. In the present study, we describe the decellularization of porcine livers to generate liver constructs at a scale that can be clinically relevant. Adult ischemic porcine livers were successfully decellularized using a customized perfusion protocol, the decellularization process preserved the ultrastructural extracellular matrix components, functional characteristics of the native microvascular and the bile drainage network of the liver, and growth factors necessary for angiogenesis and liver regeneration. Furthermore, isolated hepatocytes engrafted and reorganized in the porcine decellularized livers using a human-sized organ culture system. These results provide proof-of-principle for the generation of a human-sized, three-dimensional organ scaffold as a potential structure for human liver grafts reconstruction for transplantation to treat liver disease.

The influence of donor age on liver regeneration and hepatic progenitor cell populations.

BACKGROUND Recent reports suggest that donor age might have a major impact on recipient outcome in adult living donor liver transplantation (LDLT), but the reasons underlying this effect remain unclear. The aims of this study were to compare liver regeneration between young and aged living donors and to evaluate the number of Thy-1+ cells, which have been reported to be human hepatic progenitor cells. METHODS LDLT donors were divided into 2 groups (Group O, donor age ≥ 50 years, n = 6 and Group Y, donor age ≤ 30 years, n = 9). The remnant liver regeneration rates were calculated on the basis of computed tomography volumetry on postoperative days 7 and 30. Liver tissue samples were obtained from donors undergoing routine liver biopsy or patients undergoing partial hepatectomy for metastatic liver tumors. Thy-1+ cells were isolated and counted using immunomagnetic activated cell sorting (MACS) technique. RESULTS Donor liver regeneration rates were significantly higher in young donors compared to old donors (P = .042) on postoperative day 7. Regeneration rates were significantly higher after right lobe resection compared to rates after left lobe resection. The MACS findings showed that the number of Thy-1+ cells in the human liver consistently tended to decline with age. CONCLUSION Our study revealed that liver regeneration is impaired with age after donor hepatectomy, especially after right lobe resection. The declining hepatic progenitor cell population might be one of the reasons for impaired liver regeneration in aged donors.

International Survey on Technical Aspects of Laparoscopic Liver Resection: a web-based study on the global diffusion of laparoscopic liver surgery prior to the 2nd International Consensus Conference on Laparoscopic Liver Resection in Iwate, Japan.

The technique of laparoscopic liver resection (LLR) has been greatly improved since the first international consensus conference. Our aim was to evaluate the worldwide spread of LLR prior to the 2nd International Consensus Conference on Laparoscopic Liver Resection in Iwate, Japan (4–6 October 2014). The International Survey on Technical Aspects of Laparoscopic Liver resection was designed to assess dissemination of LLR, indications, and the surgical techniques. The anonymous questionnaire was e‐mailed to liver surgeons worldwide. A total of 448 liver surgeons responded to the survey. The peak age range of surgeons performing LLR was 41–50 years. Japan had by far the largest number of respondents (n = 223), followed by the US (n = 38) and France (n = 20). In Japan, the majority of surgeons performing LLR belonged to community hospitals, where LLR has been increasingly used since its implementation in 2009 or later, comprising up to 40% of all liver resection cases. In contrast, in North America and Europe, LLR was mostly performed at academic medical centers. LLR has undergone global dissemination after the first international consensus conference in 2008. Japan has experienced unparalleled, explosive diffusion characterized by the adoption of LLR at middle‐tier, regional institutions.

Mesenchymal stem cells support hepatocyte function in engineered liver grafts

Recent studies suggest that organ decellularization is a promising approach to facilitate the clinical application of regenerative therapy by providing a platform for organ engineering. This unique strategy uses native matrices to act as a reservoir for the functional cells which may show therapeutic potential when implanted into the body. Appropriate cell sources for artificial livers have been debated for some time. The desired cell type in artificial livers is primary hepatocytes, but in addition, other supportive cells may facilitate this stem cell technology. In this context, the use of mesenchymal stem cells (MSC) is an option meeting the criteria for therapeutic organ engineering. Ideally, supportive cells are required to (1) reduce the hepatic cell mass needed in an engineered liver by enhancing hepatocyte function, (2) modulate hepatic regeneration in a paracrine fashion or by direct contact, and (3) enhance the preservability of parenchymal cells during storage. Here, we describe enhanced hepatic function achieved using a strategy of sequential infusion of cells and illustrate the advantages of co-cultivating bone marrow-derived MSCs with primary hepatocytes in the engineered whole-liver scaffold. These co-recellularized liver scaffolds colonized by MSCs and hepatocytes were transplanted into live animals. After blood flow was established, we show that expression of adhesion molecules and proangiogenic factors was upregulated in the graft.

Correlation of postoperative recurrence in hepatocellular carcinoma with demethylation of repetitive sequences

Restriction landmark genomic scanning (RLGS) was utilized to identify novel genomic alterations in hepatocellular carcinoma (HCC). Thirty-one HCC samples were examined by RLGS. Two high intensity spots were common to several RLGS profiles of different HCCs. Nucleotide sequencing and homology search analysis showed that these spots represented repetitive sequences, Human tandem repeat sequence (Genbank, L09552) and centromeric NotI cluster (Genbank, Y10752). These intensified signals were attributable to the occurrence of demethylated areas in the recognition sequence of the NotI site of the corresponding fragments. The intensity of these spots in the RLGS profile reflects their degree of demethylation, which was significantly correlated with postoperative recurrence, even in patients regarded as belonging to the good prognosis group by conventional prognostic factors. Multivariate analysis showed that the intensities of the two spots retained independent prognostic value. This is a new type of predictive factor for HCC based on epigenetic changes in hepatocarcinogenesis, and in the future it is expected to be of great value in making preoperative diagnosis and selecting postoperative therapy.

Phase I pilot study of Wilms tumor gene 1 peptide‐pulsed dendritic cell vaccination combined with gemcitabine in pancreatic cancer

This study aimed to evaluate the feasibility of and immune response to Wilms tumor gene 1 (WT1) peptide‐pulsed dendritic cell vaccination combined with gemcitabine (DCGEM) as a first‐line therapy among patients with advanced pancreatic cancer. Ten HLA‐A*2402 patients were treated with WT1 peptide‐pulsed DC vaccination (1 × 107 cells) on days 8 and 22 and gemcitabine (1000 mg/m2) on days 1, 8 and 15. Induction of a WT1‐specific immune response was evaluated using the delayed‐type hypersensitivity (DTH) skin test, interferon‐γ enzyme‐linked immunospot and HLA tetramer assays, along with assays for various immunological factors. DCGEM was well‐tolerated, and the relative dose intensity of gemcitabine was 87%. Disease control associated with a low neutrophil/lymphocyte ratio was observed in all three patients with DTH positivity; it was also correlated with a low percentage of granulocytic myeloid derived suppressor cells in the pretreatment peripheral blood (P = 0.017). Patients with liver metastases and high levels of inflammatory markers such as C‐reactive protein and interleukin‐8 (IL‐8) showed poor survival even though a WT1‐specific immune response was induced in them. WT1 peptide‐pulsed DCGEM is feasible and effective for inducing anti‐tumor T‐cell responses. Our results support future investigations for pancreatic cancer patients with non‐liver metastases and favorable immunological conditions. This trial was registered with the University hospital Medical Information Network (UMIN) Clinical Trials Registry (http://www.umin.ac.jp/ctr/ number: UMIN‐000004855).

Abstract 1321: A novel prevention method against re-obstruction of titanium alloy stent for biliary malignancy using generation of hydroxyl radical under ultrasonic irradiation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL [introduction] Palliative biliary drainage using a metallic stent is performed for biliary obstruction by unresectable biliary carcinoma.The most important problem of biliary stenting therapy is re-obstruction due to tumor ingrowth. The generation of hydroxyl radical was investigated during ultrasonic irradiation and in the presence of TiO2(Shimizu, etal.Ultrason.Sonochem.2008).The metal stent which is used for biliary drainage is made of titanium alloy, and it is thought that it could generate hydroxyl radicals under ultrasonic irradiation in the same way. The object of this study was to establish a novel prevention method of re-obstruction of the stent using generation of hydroxyl radical under ultrasonic irradiation. [method] {in vitro}Nickel titanium alloy was ultrasonically irradiated at frequency of 1MHz for 30 seconds and the amount of generated hydroxyl radical was measured when intensity was changed to 0.1-0.5 W/cm2 by APF method. To evaluate the cytotoxic effect of the generated radicals, trypan blue exclusion assay was used.The number of living cells were counted. {in vivo}A stent re-obstruction model was established.Nude mice(BALB c nu/nu) were given an injection of 1.0×107 of human squamous carcinoma A431 cells on the back.After Seven days, a stent was put in the neighborhood of tumor.The irradiation group(n=3) was given 10 minutes-ultrasonic irradiation of 0.3W /cm2 once a day.The non-irradiation group(n=3) was not irradiated. Tumors with stents were resected on the 14th day. Tumor ingrowth area was measured and histlogical examination of the tumors was performed. [result] {in vitro}The maximum amount of hydroxyl radical generation was measured under ultrasonic irradiation of 0.3W /cm2. The number of living cells with titanium alloy under ultrasonic irradiation(0.3W /cm2 30seconds) was 79% of that without ultrasonic irradiation. {in vivo}On the 14th day, tumor ingrowth to the stent was seen in the non-irradiation group of the stent re-obstruction model.Tumor ingrowth to the stent was clearly inhibited in the irradiation group. [conclusion] Hydroxyl radical was generated from a titanium alloy stent by ultrasonic irradiation and seemed to have therapeutic efficacy. Tumor ingrowth was inhibited, and preventive effect against re-obstruction was suggested. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1321. doi:1538-7445.AM2012-1321

Epidermoid cyst originating from an intrapancreatic accessory spleen

We report a rare case of an epidermoid cyst originating from an intrapancreatic accessory spleen, in a 40-year-old Japanese man with no clinical symptoms. A cystic tumor in the pancreatic tail was detected incidentally by abdominal ultrasonography. The patient was referred to the KKR Tachikawa Hospital for further examination of the tumor. Preoperative imaging findings suggested that the tumor was an epidermoid cyst originating from an intrapancreatic accessory spleen. On both pre-and post-contrast computed tomography and magnetic resonance images, the solid compartment of the tumor had the same X-ray attenuation and intensity as the spleen. Upon surgical excision, the mass consisted of solid and cystic components that were macroscopically evident on the preoperative images. Microscopic analysis revealed that the solid component was an accessory spleen in the pancreatic tail, whereas the cystic component was lined with stratified epithelium representative of an epidermoid cyst. This is the thirteenth report (in English) of an epidermoid cyst originating from an intrapancreatic accessory spleen, and the first case to be diagnosed prior to surgery.

Laparoscopic resection of an epidermoid cyst originating from an intrapancreatic accessory spleen: Report of a case

A 67-year-old man underwent an investigation of epigastric pain and weight loss. Preoperative imaging findings suggested the presence of a tumor, which developed as an epidermoid cyst and originated from an intrapancreatic accessory spleen; however, the possibility of malignancy could not be ruled out. We therefore performed a laparoscopic-assisted distal pancreatectomy with a splenectomy for both diagnostic and treatment purposes. Five laparoscopic ports were created. After the spleen and pancreatic tail were dissected from the retroperitoneum laparoscopically, they were pulled out through a 7-cm left subcostal incision and resected with an endoscopic linear stapler. The operative time was 227 min and the blood loss was 400 ml. The postoperative course was uneventful. The final pathological diagnosis was in agreement with the preoperative diagnosis. This case demonstrates that the minimally invasive approach of laparoscopic surgery can be used safely and successfully for difficult-to-diagnose pancreatic tumors. This is the first report describing a laparoscopic resection of an epidermoid cyst originating from an intrapancreatic accessory spleen.

A new predictive factor for hepatocellular carcinoma based on two-dimensional electrophoresis of genomic DNA

Molecular genetic analyses have clarified that accumulation of genomic changes provides important steps in carcinogenesis and have identified a number of valuable genetic markers for certain cancers. To date, however, no prognostic markers have been identified for hepatocellular carcinoma (HCC). In this study, we used restriction landmark genomic scanning (RLGS), a new high-speed screening method for multiple genomic changes, to detect unknown genetic alterations in HCC. Thirty-one HCC samples and their normal counterparts were examined by RLGS. Eight spot changes were common in several cases, and all were seen only on the HCC profile. Five of these spots were detected in more than 12 of 31 cases (38.7%). Viral infection had no influence on changes in the RLGS spots. The disease-free survival rate for patients with ⩾16 changed RLGS spots was significantly lower than that for patients with fewer changed RLGS spots (⩽15 spots) (P<0.001). In multivariate analysis, the number of changed spots was proven to retain an independent prognostic value (relative risk 1.095: P=0.0031). These results suggest that the number of changed RLGS spots may be a useful biological marker for recurrence of HCC.