Naoto Koike

Vascularized and functional human liver from an iPSC-derived organ bud transplant

A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver-specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine.

BENEFITS OF ADJUVANT RADIOTHERAPY AFTER RADICAL RESECTION OF LOCALLY ADVANCED MAIN HEPATIC DUCT CARCINOMA

PURPOSE The objective of this study was to determine the benefits of adjuvant radiotherapy after radical resection of locally advanced main hepatic duct carcinoma (Klatskin tumor). METHODS AND MATERIALS We conducted a retrospective review of 63 patients who underwent surgical resection of Stage IVA Klatskin tumor. Of the 63 patients, 47 had microscopic tumor residue (RT1). Twenty-eight of the 47 patients with RT1 were treated by adjuvant radiotherapy and the remaining 19 patients were treated exclusively by surgical resection. Seventeen of the 28 patients with RT1 were treated by both intraoperative radiotherapy (IORT) and postoperative radiotherapy (PORT); of the remaining 11 patients with RT1, 6 underwent resection and IORT, and 5 underwent resection and PORT. RESULTS The major complication and 30-day operative death rates were significantly lower in the radiation group (9.5% and 0.0%, respectively) than in the resection alone group (28.5% and 9.5%, respectively). Of the eight 5-year survivors with RT1, 6 had adjuvant radiotherapy and the remaining 2 had resection alone. Adjuvant radiotherapy for patients with RT1 yielded significantly (p = 0.0141) higher 5-year survival rates (33.9%) than in the resection alone group (13.5 %). The best 5-year survival rate (39.2 %) was found in patients who underwent a combination of IORT and PORT after resection. The local-regional control rate was significantly higher in the adjuvant radiation group than in the resection alone group (79.2% vs. 31.2%). CONCLUSION Our data clearly suggest the improved prognosis of patients with locally advanced Klatskin tumor by integrated adjuvant radiotherapy with IORT and PORT to complete gross tumor resection with acceptable treatment mortality and morbidity.

Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant

Generation of functional and vascularized organs from human induced pluripotent stem cells (iPSCs) will facilitate our understanding of human developmental biology and disease modeling, hopefully offering a drug-screening platform and providing novel therapies against end-stage organ failure. Here we describe a protocol for the in vitro generation of a 3D liver bud from human iPSC cultures and the monitoring of further hepatic maturation after transplantation at various ectopic sites. iPSC-derived specified hepatic cells are dissociated and suspended with endothelial cells and mesenchymal stem cells. These mixed cells are then plated onto a presolidified matrix, and they form a 3D spherical tissue mass termed a liver bud (iPSC-LB) in 1–2 d. To facilitate additional maturation, 4-d-old iPSC-LBs are transplanted in the immunodeficient mouse. Live imaging has identified functional blood perfusion into the preformed human vascular networks. Functional analyses show the appearance of multiple hepatic functions in a chronological manner in vivo.

Expression of TNF-related apoptosis-inducing ligand (TRAIL) and its receptors in gastric carcinoma and tumor-infiltrating lymphocytes: a possible mechanism of immune evasion of the tumor

Abstract.Purpose: TNF-related apoptosis-inducing ligand (TRAIL) and its receptors have recently been known to be responsible for apoptotic signaling molecules in tumor cell lines and tissues. These molecules have been reported to be expressed on merely a transcription level, but not on a protein level. Moreover, little is known about TRAIL-mediated apoptosis in human carcinoma in vivo. Methods: We investigated the presence and functional status of TRAIL and its receptors, DR4, DR5, and DcR2 on tumor as well as tumor-infiltrating lymphocytes (TIL) in primary (n=37), and metastatic gastric carcinoma from malignant ascites (n=37) by a flow cytometry. In addition, phenotypic proportions of major T-cell subsets or B-cells in TIL were also determined. Results: Membrane-bound TRAIL/its receptors are constitutively expressed at high levels in primary and metastatic carcinomas in nearly all the patients. Apoptotic tumor cells detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick and labeling (TUNEL) were barely identified in primary and metastatic carcinomas. TIL in primary carcinoma showed a very low level of expression of TRAIL/its receptors and TUNEL-positive cells. In metastatic carcinoma, however, there was significant overexpression of TRAIL/its receptors in TIL associated with a higher frequency of apoptotic cell death detected by TUNEL. The TIL within metastatic carcinoma, but not within primary carcinoma, revealed the increased proportions of CD3+ T cells bearing CD8+CD11b–, CD8+CD11b+, and CD4+CD62L–, CD4+CD62L+ surface phenotype in patients. Conclusions: These results suggest that TRAIL+ and DcR2+ metastatic carcinoma from malignant ascites could not only have resistance to DR4/DR5-induced apoptosis, but also might take the TRAIL-mediated counterattack against activated CD3+ T cells. These functions of the cancer cells would neutralize host immune responses at the effector phase, and accelerate further invasion and/or metastasis of carcinoma through the escape from immune attack.

Fas receptor counterattack against tumor-infiltrating lymphocytes in vivo as a mechanism of immune escape in gastric carcinoma.

Abstract We investigated the presence and functional status of surface expression of the Fas receptor (FasR) and its ligand (FasL) in tumor and tumor-infiltrating lymphocytes (TIL) in gastric carcinoma (n=36) from the primary locus, metastatic gastric carcinoma (n=30) from malignant ascites, and benign gastric mucosa (n=30) for the control. The quantitative analysis was based on the percentage of positive cells by a flow cytometry. The results showed that the membrane-bound FasL molecule was constitutively expressed in primary and metastatic gastric carcinomas as well as normal gastric epithelium in nearly all the patients. In particular, metastatic carcinoma proved to aberrantly express the FasL molecule. On the other hand, FasR expression ranged from minimal or absent in primary and metastatic gastric carcinomas, suggesting that the carcinoma might be rendered less sensitive toward FasR-induced killing. Apoptotic tumor cells detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick and labeling (TUNEL) were barely identified in primary and metastatic carcinomas. In the analysis of TIL, the expression of FasR and FasL, and apoptotic TIL could not usually be observed in primary gastric carcinoma. In metastatic carcinoma, however, there was significant overexpression of FasR and FasL in immune TIL associated with a higher frequency of apoptotic cell death detected by TUNEL. The results suggest that metastatic carcinoma expressing FasL, but not FasL+ primary carcinoma, might evade the immune attack by apoptotic depletion of activated TIL through the FasR/FasL systems. These results provide the direct and quantitative evidence of FasR counterattacks and/or paracrine fratricides as a mechanism of tumor-immune escape in vivo in human cancer.

Expression of cyclooxygenase-2 in the subserosal layer correlates with postsurgical prognosis of pathological tumor stage 2 carcinoma of the gallbladder.

Postsurgical recurrence at distant sites frequently occurs in pathological tumor stage 2 (pT2) carcinoma of the gallbladder even though the carcinoma is limited to the gallbladder wall. Little is known, however, about the molecular events leading to its development and progression. A large body of evidence suggests that cyclooxygenase‐2 (COX‐2) is up‐regulated in carcinoma tissues and plays roles in promoting cell‐proliferation, growth and metastasis of carcinoma cells. In the present study, immunohistochemistry was performed to determine the expression levels of COX‐2 in the subserosal layer of 33 cases of pT2 gallbladder carcinoma in which curative resections had been performed and to determine the correlations of the expression levels of COX‐2 with mode of recurrence and postsurgical survival. Immunostaining of COX‐2 in the epithelia was recognized in more than 80% of normal epithelia, noncancerous pathological lesions of the gallbladder except for intestinal metaplasia and pT1–4 carcinoma specimens. Intense staining was observed in large percentages of hyperplastic lesions (65%), pT2 carcinoma specimens (76%) and pT3 and pT4 carcinoma specimens (64%) compared to the percentages of normal epithelia and other pathological lesions (0–25%). Intense staining was also observed in the adjacent stroma in pT2 carcinoma specimens (33%) and in those in pT3 and pT4 carcinoma specimens (43%) but only in small percentages of the stroma adjacent to normal epithelia and pathological lesions (0–8%). In situ hybridization confirmed the existence of COX‐2 mRNA in both the cancerous epithelia and adjacent stroma of pT2‐pT4 carcinomas. In 33 cases of pT2 carcinoma, distant recurrence, i.e., liver metastasis, was seen in 3 of 9 cases of pT2 carcinoma (33%, P<0.05) with intense stromal staining in the subserosal layer and in 1 of 24 cases (4%) without intense staining, whereas no significant correlation was found between parameters of pathological malignancies (histological grade, lymphatic permeation, venous permeation and lymph node metastasis) and the intensity of stromal staining in the subserosal layer. The postsurgical survival outcome was significantly poorer in the former than in the latter (p = 0.010). In pT2 gallbladder carcinoma, upregulation of COX‐2 in the stroma adjacent to the cancerous epithelia in the subserosal layer correlates with the aggressiveness of the disease, such as the tendency to form distant recurrences. This phenotype may serve as a unique biological feature associated with the malignant behavior of pT2 gallbladder carcinoma.

Invasive potentials of gastric carcinoma cell lines: Role of α2 and α6 integrins in invasion

The potentials of the two major histological types of gastric carcinoma to invade through extracellular matrices were studied with cell lines. We found that the invasive potential of intestinal-type carcinoma cells (MKN-28 and MKN-74) were higher than those of diffuse-type carcinoma cells (MKN-45 and KATO-III). To investigate whether the α2 and α6 integrin adhesion molecules are responsible for, or involved in carcinoma invasion, we further studied α2 and α6 expression patterns in these two types of cell line. Although fluorescence-activated cell sorting analysis revealed that all cells examined invariably expressed these integrin molecules, their expressional patterns were different among different cell lines. The intestinal-type carcinoma cells expressed integrins mainly along the cell-cell contact region, whereas the diffuse-type carcinoma cells showed a diffuse cytoplasmic pattern of integrin expression. Invasion by MKN-28, MKN-74 and MKN-45 cells through reconstituted basement membrane or type I collagen gel was significantly inhibited (P<0.05) by 50 μg/ml anti-(α2 integrin) or anti-(α6 integrin) monoclonal antibodies. Our results suggest that active invasiveness is stronger in the intestinal-type than in the diffuse-type carcinoma cells and that α2 and α6 integrins play important roles in invasion of both types of gastric carcinoma cell lines.

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.

Generation of functional human vascular network.

BACKGROUND One of the major obstacles in regenerating thick, complex tissues such as the liver is their need for vascularization, which is essential to maintain cell viability during tissue growth and to induce structural organization. Herein, we have described a method to engineer a functional human vascular network. METHODS Enhanced green fluorescence protein-labeled human umbilical vein endothelial cells (GFP-HUVECs) were cocultivated with kusabira orange-labeled human mesenchymal stem cells (KO-hMSCs) inside a collagen/fibronectin matrix. Premature vascular network formation was visualized by fluorescence microscopy imaging. Furthermore, constructs prevascularized in vitro were implanted into a transparency window in immunodeficient mice. RESULTS Following several days of cultivation, GFP-HUVECs formed vessel-like structures that were stabilized by pericytes differentiated from KO-hMSCs. After implantation in vivo, the patency of human vascular structures was proved by rhodamine dextran infusion. These functional vascular structures remained for over 2 months. DISCUSSION Vascularization is the key challenge to organ generation. We successfully generated human vascular networks inside a matrix. Integration of parenchymal cells using our engineering technique should facilitate future efforts to reconstitute vascularized human organ systems in vitro.

Engineering of human hepatic tissue with functional vascular networks

Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.