Cotransplantation With Adipose Tissue-derived Stem Cells Improves Engraftment of Transplanted Hepatocytes.

BACKGROUND: Hepatocyte transplantation is expected to be an alternative therapy to liver transplantation; however, poor engraftment is a severe obstacle to be overcome. The adipose tissue-derived stem cells (ADSCs) are known to improve engraftment of transplanted pancreatic islets, which have many similarities to the hepatocytes. Therefore, we examined the effects and underlying mechanisms of ADSC cotransplantation on hepatocyte engraftment. METHODS: Hepatocytes and ADSCs were cotransplanted into the renal subcapsular space and livers of syngeneic analbuminemic rats, and the serum albumin level was quantified to evaluate engraftment. Immunohistochemical staining and fluorescent staining to trace transplanted cells in the liver were also performed. To investigate the mechanisms, cocultured supernatants were analyzed by a multiplex assay and inhibition test using neutralizing antibodies for target factors. RESULTS: Hepatocyte engraftment at both transplant sites was significantly improved by ADSC cotransplantation ( P < 0.001, P < 0.001). In the renal subcapsular model, close proximity between hepatocytes and ADSCs was necessary to exert this effect. Unexpectedly, ≈50% of transplanted hepatocytes were attached by ADSCs in the liver. In an in vitro study, the hepatocyte function was significantly improved by ADSC coculture supernatant ( P < 0.001). The multiplex assay and inhibition test demonstrated that hepatocyte growth factor, vascular endothelial growth factor, and interleukin-6 may be key factors for the abovementioned effects of ADSCs. CONCLUSIONS: The present study revealed that ADSC cotransplantation can improve the engraftment of transplanted hepatocytes. This effect may be based on crucial factors, such as hepatocyte growth factor, vascular endothelial growth factor, and interleukin-6, which are secreted by ADSCs.

Short-term inhalation of isoflurane improves the outcomes of intraportal hepatocyte transplantation.

Clinical hepatocyte transplantation (HTx) is only performed without general anesthesia, while inhalation anesthetics are usually used in animal experiments. We hypothesized that isoflurane may be a possible reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx. Syngeneic rat hepatocytes (1.0 × 107) were transplanted to analbuminemic rats with (ISO group) and without (AW group) isoflurane. The serum albumin, AST, ALT, LDH levels and several inflammatory mediators were analyzed. Immunohistochemical staining and ex vivo imaging were also performed. The serum albumin levels of the ISO group were significantly higher in comparison to the AW group (p < 0.05). The serum AST, ALT, LDH levels of the ISO group were significantly suppressed in comparison to the AW group (p < 0.0001, respectively). The serum IL-1ß, IL-10, IL-18, MCP-1, RNTES, Fractalkine and LIX levels were significantly suppressed in the ISO group. The ischemic regions of the recipient livers in the ISO group tended to be smaller than the AW group; however, the distribution of transplanted hepatocytes in the liver parenchyma was comparable between the two groups. Isoflurane may at least in part be a reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx.

Preferable Transplant Site for Hepatocyte Transplantation in a Rat Model.

Intraportal injection is regarded as the current standard procedure of hepatocyte transplantation (HTx). In islet transplantation, which shares many aspects with HTx, recent studies have clarified that instant blood-mediated inflammatory reaction (IBMIR), characterized by strong innate immune responses, can cause poor engraftment, so other transplant sites to avoid such a reaction have been established. Although IBMIR was reported to occur in HTx, few reports have evaluated alternative transplant sites for HTx. In this study, we sought to determine the optimum transplant site for HTx. Rat hepatocytes (1.0 × 107) were transplanted at the 9 transplant sites (intraportal (IPO), intrasplenic (IS), liver parenchyma, subcutaneous, intraperitoneal, renal subcapsular, muscle, inguinal subcutaneous white adipose tissue, and omentum) of analbuminemic rats. The serum albumin levels, immunohistochemical staining (albumin, TUNEL, and BrdU), and in vivo imaging of the grafts were evaluated. The serum albumin levels of the IPO group were significantly higher than those of the other groups (p < .0001). The BrdU-positive hepatocyte ratio of liver in the IS group (0.9% ± 0.2%) was comparable to that of the IPO group (0.9% ± 0.3%) and tended to be higher than that of the spleen in the IS group (0.5% ± 0.1%, p = .16). Considering the in vivo imaging evaluation and the influence of splenectomy, the graft function in the IS group may be almost entirely achieved by hepatocytes that have migrated to the liver. The present study clearly showed that the intraportal injection procedure is more efficient than other procedures for performing HTx.

The Liver Surface Is an Attractive Transplant Site for Pancreatic Islet Transplantation.

In the current clinical islet transplantation, intraportal transplantation is regarded as the gold-standard procedure. However, in this procedure, 50 to 70% of the transplanted islets are immediately damaged due to a strong innate immune response based on islet-blood contact. We investigated the transplant efficiency of a novel method of liver surface transplantation using a syngeneic keratinocyte sheet to avoid islet-blood contact. To examine the influence of the keratinocyte sheet, substantial amounts of syngeneic islets (8 IEQs/g) were transplanted on the liver surface of diabetic rats, while marginal amounts of islets (4 IEQs/g) were transplanted via intraportal transplantation to compare the transplant efficiency. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, and in vivo imaging findings of the cell sheet were evaluated. The study showed that islet transplantation to the liver surface immediately followed by a syngeneic keratinocyte sheet covering was effective for curing diabetic rats, while no rats were cured in the group without the cell sheet. Notably, islet grafts transplanted via this approach appeared to penetrate into the liver parenchyma. However, the transplant efficiency did not reach that of intraportal transplantation. Further refinements of this approach by introducing mesothelial or fibroblast cell sheets in combination with a preferable scaffold for islet grafts may help to improve the transplant efficiency.

Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model.

Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler's disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from Idua knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neor) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neor sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neor removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients.

A Simple and Useful Predictive Assay for Evaluating the Quality of Isolated Hepatocytes for Hepatocyte Transplantation.

No optimal assay for assessing isolated hepatocytes before hepatocyte transplantation (HTx) has been established, therefore reliable and rapid assays are warranted. Isolated rat hepatocytes were dipped in a water bath (necrosis model), and were also cultured with Okadaic acid (apoptosis model) or vehicle, followed by cellular assessment including trypan blue exclusion (TBE) viability, ADP /ATP ratio, plating efficiency (PE), DNA quantity and ammonia elimination. Hepatocytes were transplanted into the liver of analbuminemic rats, subsequently engraftment was assessed by serum albumin and the histology of transplanted grafts. In the necrosis model, the ADP/ATP ratio was strongly and negatively correlated with the TBE (R2 = 0.559, P < 0.001). In the apoptosis model, the ADP/ATP ratio assay, PE, DNA quantification and an ammonia elimination test clearly distinguished the groups (P < 0.001, respectively). The ADP/ATP ratio, PE and DNA quantity were well-correlated and the ammonia elimination was slightly correlated with the transplant outcome. TBE could not distinguish the groups and was not correlated with the outcome. The ADP/ATP ratio assay predicted the transplant outcome. PE and DNA quantification may improve the accuracy of the retrospective (evaluations require several days) quality assessment of hepatocytes. The ADP/ATP ratio assay, alone or with a short-term metabolic assay could improve the efficiency of HTx.

Human iPS Cell-based Liver-like Tissue Engineering at Extrahepatic Sites in Mice as a New Cell Therapy for Hemophilia B.

Instead of liver transplantation or liver-directed gene therapy, genetic liver diseases are expected to be treated effectively using liver tissue engineering technology. Hepatocyte-like cells (HLCs) generated from human-induced pluripotent stem (iPS) cells are an attractive unlimited cell source for liver-like tissue engineering. In this study, we attempted to show the effectiveness of human iPS cell-based liver-like tissue engineering at an extrahepatic site for treatment of hemophilia B, also called factor IX (FIX) deficiency. HLCs were transplanted under the kidney capsule where the transplanted cells could be efficiently engrafted. Ten weeks after the transplantation, human albumin (253 µg/mL) and α-1 antitrypsin (1.2 µg/mL) could be detected in the serum of transplanted mice. HLCs were transplanted under the kidney capsule of FIX-deficient mice. The clotting activities in the transplanted mice were approximately 5% of those in wild-type mice. The bleeding time in transplanted mice was shorter than that in the nontransplanted mice. Taken together, these results indicate the success in generating functional liver-like tissues under the kidney capsule by using human iPS cell-derived HLCs. We also demonstrated that the human iPS cell-based liver-like tissue engineering technology would be an effective treatment of genetic liver disease including hemophilia B.

Adult hepatocytes direct liver organogenesis through non-parenchymal cell recruitment in the kidney.

BACKGROUND & AIMS: Since the first account of the myth of Prometheus, the amazing regenerative capacity of the liver has fascinated researchers because of its enormous medical potential. Liver regeneration is promoted by multiple types of liver cells, including hepatocytes and liver non-parenchymal cells (NPCs), through complex intercellular signaling. However, the mechanism of liver organogenesis, especially the role of adult hepatocytes at ectopic sites, remains unknown. In this study, we demonstrate that hepatocytes alone spurred liver organogenesis to form an organ-sized complex 3D liver that exhibited native liver architecture and functions in the kidneys of mice. METHODS: Isolated hepatocytes were transplanted under the kidney capsule of monocrotaline (MCT) and partial hepatectomy (PHx)-treated mice. To determine the origin of NPCs in neo-livers, hepatocytes were transplanted into MCT/PHx-treated green fluorescent protein transgenic mice or wild-type mice transplanted with bone marrow cells isolated from green fluorescent protein-mice. RESULTS: Hepatocytes engrafted at the subrenal space of mice underwent continuous growth in response to a chronic hepatic injury in the native liver. More than 1.5 years later, whole organ-sized liver tissues with greater mass than those of the injured native liver had formed. Most remarkably, we revealed that at least three types of NPCs with similar phenotypic features to the liver NPCs were recruited from the host tissues including bone marrow. The neo-livers in the kidney exhibited liver-specific functions and architectures, including sinusoidal vascular systems, zonal heterogeneity, and emergence of bile duct cells. Furthermore, the neo-livers successfully rescued the mice with lethal liver injury. CONCLUSION: Our data clearly show that adult hepatocytes play a leading role as organizer cells in liver organogenesis at ectopic sites via NPC recruitment. LAY SUMMARY: The role of adult hepatocytes at ectopic locations has not been clarified. In this study, we demonstrated that engrafted hepatocytes in the kidney proliferated, recruited non-parenchymal cells from host tissues including bone marrow, and finally created an organ-sized, complex liver system that exhibited liver-specific architectures and functions. Our results revealed previously undescribed functions of hepatocytes to direct liver organogenesis through non-parenchymal cell recruitment and organize multiple cell types into a complex 3D liver at ectopic sites. Transcript profiling: Microarray data are deposited in GEO (GEO accession: GSE99141).

Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes.

Gene therapy during neonatal and infant stages is a promising approach for hemophilia B, a congenital disorder caused by deficiency of blood coagulation factor IX (FIX). An adenovirus (Ad) vector has high potential for use in neonatal or infant gene therapy for hemophilia B due to its superior transduction properties; however, leaky expression of Ad genes often reduces the transduction efficiencies by Ad protein-mediated tissue damage. Here, we used a novel Ad vector, Ad-E4-122aT, which exhibits a reduction in the leaky expression of Ad genes in liver, in gene therapy studies for neonatal hemophilia B mice. Ad-E4-122aT exhibited significantly higher transduction efficiencies than a conventional Ad vector in neonatal mice. In neonatal hemophilia B mice, a single neonatal injection of Ad-E4-122aT expressing human FIX (hFIX) (Ad-E4-122aT-AHAFIX) maintained more than 6% of the normal plasma hFIX activity levels for approximately 100 days. Sequential administration of Ad-E4-122aT-AHAFIX resulted in more than 100% of the plasma hFIX activity levels for more than 100 days and rescued the bleeding phenotypes of hemophilia B mice. In addition, immunotolerance to hFIX was induced by Ad-E4-122aT-AHAFIX administration in neonatal hemophilia B mice. These results indicated that Ad-E4-122aT is a promising gene delivery vector for neonatal or infant gene therapy for hemophilia B.

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation.

BACKGROUND: No optimal methods for short-term hepatocyte preservation have been established. We have recently developed a prominent oxygen-permeable bag (Tohoku Device [TD]) for pancreatic islet culture and transplantation. In this study, we investigated whether TD is also effective for hepatocyte preservation and tried to optimize other conditions. METHODS: Hepatocytes were preserved in the following conditions, and their outcomes were observed. First, the effectiveness of TD was investigated. Second, hepatocyte medium (HM) and organ preservation solutions with or without fetal bovine serum (FBS) were compared. Third, as supplementations, FBS and human serum albumin (HSA) were compared. Fourth, low, room and high temperature were compared. And finally, hepatocytes preserved in various conditions were transplanted into the subrenal capsule space of nonalbumin rats and engrafted areas were assessed. RESULTS: The survival rate of hepatocytes preserved in TD tended to be higher and their viability and function were maintained significantly greater than those of non-TD group. Irrespective of FBS supplementation, the survival rate of HM group was significantly higher than those of organ preservation solution group while viabilities and plating efficiency were similar among them. Although survival rates of groups without FBS were extremely low, results of HSA supplemented group were not inferior to FBS supplemented group. Hepatocytes preserved at high temperature had the worst results. The engrafted area of TD group tended to be higher than those of other groups. CONCLUSIONS: TD is effective for short-term hepatocyte preservation. HSA is a useful substitute for FBS, and preserving in HM at low temperature is recommended.

Fabrication of functional 3D hepatic tissues with polarized hepatocytes by stacking endothelial cell sheets in vitro.

Cell sheet stratification technology has been used for reconstituting highly functional three-dimensional (3D) hepatic tissues in vitro. Triple-layered hepatic tissues with a hepatocyte-specific polarity were fabricated by sandwiching a hepatocyte sheet (Hep sheet) between two endothelial cell (EC) sheets. The morphological and functional characteristics of the triple-layered hepatic construct (EC-Hep-EC) were evaluated and compared with those of a double-layered hepatic construct with a single EC sheet (Hep-EC) and a Hep sheet only. Transmission electron microscope (TEM) observations revealed that the extracellular matrix was observed to be deposited in the space between the ECs and hepatocytes on both the upper and lower sides of the hepatocytes in the EC-Hep-EC construct. Immunohistochemistry with basolateral (CD147) and apical [multidrug resistance-associated protein (MRP2)] membrane polarity markers clearly showed the recovery of in vivo-like hepatocyte polarization in the EC-Hep-EC group. In addition, hepatocyte-specific functions, including albumin secretion, ammonia removal and the induction of cytochrome P450, were also highly preserved. The presented technology for stratifying multiple cell sheets was simple in operation and successfully reproduced both the heterotypic/homotypic cell-cell and cell-matrix interactions with the inherent hepatocyte configurations, thus closely mimicking the in vivo environment. The triple-layered 3D hepatic constructs could therefore be valuable as a new experiment tool for drug-screening tests, an implantable tissue model for cell-based therapies and an efficient culture platform for bioartificial liver devices. Copyright © 2015 John Wiley & Sons, Ltd.

Transplantation of a human iPSC-derived hepatocyte sheet increases survival in mice with acute liver failure.

BACKGROUND & AIMS: Hepatocyte transplantation is one of the most attractive approaches for the treatment of patients with liver failure. Because human induced pluripotent stem cell-derived hepatocyte-like cells (iPS-HLCs) can be produced on a large scale and generated from a patient with liver failure, they are expected to be used for hepatocyte transplantation. However, when using conventional transplantation methods, i.e., intrasplenic or portal venous infusion, it is difficult to control the engraftment efficiency and avoid unexpected engraftment in other organs because the transplanted cells are delivered into blood circulation before their liver engraftment. METHODS: In this study, to resolve these issues, we attempted to employ a cell sheet engineering technology for experimental hepatocyte transplantation. The human iPS-HLC sheets were attached onto the liver surfaces of mice with liver injury. RESULTS: This method reduced unexpected engraftment in organs other than the liver compared to that by intrasplenic transplantation. Human albumin levels in the mice with human iPS-HLC sheets were significantly higher than those in the intrasplenically-transplanted mice, suggesting the high potential for cell engraftment of the sheet transplantation procedure. In addition, human iPS-HLC sheet transplantation successfully ameliorated lethal acute liver injury induced by the infusion of carbon tetrachloride (CCl4). Moreover, we found that the hepatocyte growth factor secreted from the human iPS-HLC sheet played an important role in rescuing of mice from acute hepatic failure. CONCLUSIONS: Human iPS-HLC sheet transplantation would be a useful and reliable therapeutic approach for a patient with severe liver diseases.

Prevention of alcohol-induced hyperhomocysteinemia by suppression of SHP.

Chronic alcohol consumption is a major public health problem that frequently leads to the development of liver steatosis, fibrosis, and eventually cirrhosis and hepatocellular carcinoma. Hyperhomocysteinemia is a pathological consequence of alcoholic liver disease (ALD) and is attributed to hepatic endoplasmic reticulum (ER) stress and insulin resistance. However, the regulatory function of nuclear receptors in ALD associated with dysregulation of homocysteine metabolism remains largely unknown. Nuclear receptor small heterodimer partner (SHP, NROB2) is a pleiotropic transcriptional repressor involved in regulating various metabolic path-ways in the liver. This study investigated a critical role of SHP in alcohol-induced hyperhomocysteinemia. . The expression and enzymatic activities of betaine-homocysteine S-methyltransferase (BHMT) and cystathionine y -lyase (CTH) were significantly increased in the liver of SHP- knockout (SKO) mice as compared to the wild-type mice. The substrates of BHMT and CTH, such as betaine, choline and cystathionine, were decreased in SKO liver while their products including hydrogen sulfide and cysteine were increased. However, methionine and homocysteine were not altered by SHP- deficiency, suggesting that the methionine cycle is activated in SKO mice. Forkhead box A (FOXA)- binding site was identified in both the BHMT and CTH promoters. Luciferase assay demonstrated that FOXAI, but not FOXA2, activated both BHMT and CTH promoters through the FOXA-binding site. Overexpression of FOXA1 induced BHMT and CTH expression in Hepal-6 cells, which was inhibited by SHP coexpression. Consistently, alcohol-induced hyperhomocysteinemia, and homocysteine-induced hepatic ER stress and glucose intolerance were abrogated in SKO mice. These novel findings identified SHP and FOXA1 as important regulators of hepatic homocysteine metabolism. Because hyper-homocysteinemia is a risk factor for cardiovascular disease and insulin resistance, and is often associated with ALD and metabolic syndrome, SHP and FOXA1 could be used as potential targets for hyperhomocysteinemia and its related diseases. Taken together, these results shed light on the regulatory mechanism of homocysteine metabolism in the liver.

A heparin-modified thermoresponsive surface with heparin-binding epidermal growth factor-like growth factor for maintaining hepatic functions in vitro and harvesting hepatocyte sheets.

A heparin-modified thermoresponsive surface bound with heparin-binding epidermal growth factor-like growth factor (HB-EGF) was designed to allow creation of transferrable and functional hepatocyte sheets. A heparin-modified thermoresponsive surface was prepared by covalently tethering heparin onto poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide)-grafted tissue culture polystyrene surfaces (Heparin-IC). HB-EGFs were able to stably bind to heparin-IC via affinity interaction. The survival of primary rat hepatocytes was maintained through HB-EGF-bound heparin-IC (HB-EGF/heparin-IC). Moreover, cultured rat primary hepatocytes on HB-EGF/heparin-IC exhibited higher albumin-secretion than hepatocytes cultured on PIPAAm-grafted and collagen-coated surfaces with soluble HB-EGF in the culture medium, regardless of whether soluble EGF was added. These results suggested that HB-EGF/heparin-IC is able to effectively maintain hepatic function via continuous signaling of HB-EGF. After a 4-day cultivation, the cultured hepatocytes on HB-EGF/heparin-IC detached as a cell sheet with fibronectin and HB-EGF only after the temperature was lowered to 20 °C. In addition, higher expression of hepatocyte-specific genes (albumin, hepatocyte nuclear factor 4 alpha, coagulation factor VII, and coagulation factor IX) in hepatocyte sheets was detected on HB-EGF/heparin-IC than on a PIPAAm surface with soluble HB-EGF, indicating that HB-EGF/heparin-IC suppressed the dedifferentiation of cultured hepatocytes. Hence, heparin-modified thermoresponsive surfaces bound with HB-EGF facilitate the fabrication of transferrable hepatocyte sheets with intact hepatic functions and have the potential to provide an in vitro culture system using functional hepatocyte sheet tissues, which may serve as an effective hepatocyte-based tissue engineering platform for liver disease treatments.

Human mesenchymal stem cell-engineered hepatic cell sheets accelerate liver regeneration in mice.

Mesenchymal stem cells (MSCs) are an attractive cell source for cell therapy. Based on our hypothesis that suppression of Wnt/ß-catenin signal enhances hepatic differentiation of human MSCs, we developed human mesenchymal stem cell-engineered hepatic cell sheets by a small molecule compound. Screening of 10 small molecule compounds was performed by WST assay, TCF reporter assay, and albumin mRNA expression. Consequently, hexachlorophene suppressed TCF reporter activity in time- and concentration-dependent manner. Hexachlorophene rapidly induced hepatic differentiation of human MSCs judging from expression of liver-specific genes and proteins, PAS staining, and urea production. The effect of orthotopic transplantation of human mesenchymal stem cell-engineered hepatic cell sheets against acute liver injury was examined in one-layered to three-layered cell sheets system. Transplantation of human mesenchymal stem cell-engineered hepatic cell sheets enhanced liver regeneration and suppressed liver injury. The survival rates of the mice were significantly improved. High expression of complement C3 and its downstream signals including C5a, NF-κB, and IL-6/STAT-3 pathway was observed in hepatic cell sheets-grafted tissues. Expression of phosphorylated EGFR and thioredoxin is enhanced, resulting in reduction of oxidative stress. These findings suggest that orthotopic transplantation of hepatic cell sheets manufactured from MSCs accelerates liver regeneration through complement C3, EGFR and thioredoxin.

Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice.

Subcutaneous liver tissue engineering is an attractive and minimally invasive approach used to curative treat hepatic failure and inherited liver diseases. However, graft failure occurs frequently due to insufficient infiltration of blood vessels (neoangiogenesis), while the maintenance of hepatocyte phenotype and function requires in vivo development of the complex cellular organization of the hepatic lobule. Here we describe a subcutaneous human liver construction allowing for rapidly vascularized grafts by transplanting engineered cellular sheets consisting of human primary hepatocytes adhered onto a fibroblast layer. The engineered hepatocyte/fibroblast sheets (EHFSs) showed superior expression levels of vascularization-associated growth factors (vascular endothelial growth factor, transforming growth factor beta 1, and hepatocyte growth factor) in vitro. EHFSs developed into vascularized subcutaneous human liver tissues contained glycogen stores, synthesized coagulation factor IX, and showed significantly higher synthesis rates of liver-specific proteins (albumin and alpha 1 anti-trypsin) in vivo than tissues from hepatocyte-only sheets. The present study describes a new approach for vascularized human liver organogenesis under mouse skin. This approach could prove valuable for establishing novel cell therapies for liver diseases.

Evaluation of transduction properties of an adenovirus vector in neonatal mice.

In gene therapy for congenital disorders, treatments during neonate and infant stages are promising. Replication-incompetent adenovirus (Ad) vectors have been used in gene therapy studies of genetic disorders; however, the transduction properties of Ad vectors in neonates and infants have not been fully examined. Accordingly, this study examined the properties of Ad vector-mediated transduction in neonatal mice. A first-generation Ad vector containing a cytomegalovirus (CMV) promoter-driven luciferase expression cassette was administered to neonatal mice on the second day of life via retro-orbital sinus. The highest Ad vector genome copy numbers and transgene expression were found in the neonatal liver. The neonatal heart exhibited the second highest levels of transgene expression among the organs examined. There was an approximately 1500-fold difference in the transgene expression levels between the adult liver and heart, while the neonatal liver exhibited only an approximately 30-fold higher level of transgene expression than the neonatal heart. A liver-specific promoter for firefly luciferase expression conferred a more than 100-fold higher luciferase expression in the liver relative to the other organs. No apparent hepatotoxicity was observed in neonatal mice following Ad vector administration. These findings should provide valuable information for gene therapy using Ad vectors in neonates and infants.

Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors.

To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells.

Human Laminin Isotype Coating for Creating Islet Cell Sheets.

Our experimental approach toward the development of new islet-based treatment for diabetes mellitus has been the creation of a monolayered islet cell construct (islet cell sheet), followed by its transplantation into a subcutaneous pocket. Previous studies describe rat laminin-5 (chain composition: α3, ß3, γ2) as a suitable extracellular matrix (ECM) for surfaces comprised of a coated temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). To progress toward the clinical application of this approach, the present study attempted to identify an optimal human ECM as a coating material on PIPAAm surfaces, which allowed islet cells to attach on the surfaces and subsequently to be harvested as a monolithic cell sheet. Dispersed rat islet cells were seeded onto PIPAAm dishes coated with various human laminin isotypes: human laminin (HL)-211, HL-332, HL-411, HL-511, and HL-placenta. Plating efficiency at day 1, the confluency at day 3, and glucose-stimulated insulin secretion test at day 3 were performed. The highest value of plating efficiency was found in the HL-332-PIPAAm group (83.1 ± 0.7%). The HL-332-PIPAAm group also showed the highest cellular confluency (98.6 ± 0.5%). Islet cells cultured on the HL-332-PIPAAm surfaces showed a positive response in the glucose-stimulated insulin secretion test. By reducing culture temperature from 37°C to 20°C in the HL-332-PIPAAm group, cells were able to be harvested as a monolithic islet sheet. The present study showed that HL-332 was an optimal human-derived ECM on a PIPAAm coating for preparing islet cell sheets.