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.

In vitro and in vivo fabrication of stable human hepatocyte tissue in combination with normal fibroblasts derived from donors of various ages.

In order to establish a minimally invasive and safe liver regenerative technology, a technique for fabricating liver tissue possessing a vascular network was developed by subcutaneously transplanting a cell sheet composed of primary human hepatocytes and normal fibroblasts. However, differences in fibroblast characteristics owing to donor age may threaten the stability of liver tissue regenerated via this technology. Herein we describe the influence of fibroblasts from multiple donors on the fabrication of engineered human hepatocyte tissues invitro and in vivo. Primary human hepatocytes were cultured with seven strains of fibroblasts derived from the skins of donors of various ages, ranging from a fetus (12 weeks) to the elderly (69 years). Engineered hepatocyte sheets were successfully harvested for all strains. At 2 weeks after the subcutaneous transplantation of the hepatocyte sheets into mice, the highest human albumin (hALB) serum concentration was noted in the mouse containing fibroblasts from a 12 year old (TIG-118). Since the platelet-derived growth factor subunit B (PDGFB) gene expression of TIG-118 cells was significantly higher than that in the other cells, PDGFB may be considered to play an important role in the initial subcutaneous engraftment of primary human hepatocytes. Even though hALB concentration exhibited a parabolic tendency with age, there was no statistically significant difference noted within 6-8 weeks after transplantation. The present study demonstrates that this technology can produce consistent and stable hepatocyte sheets that exhibit long-term survival and liver-specific functionality in vivo regardless of the fibroblast donor age.

Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×103 cells/cm2. Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-ß1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues.

Association of epicardial adipose tissue with serum level of cystatin C in type 2 diabetes.

OBJECTIVE: Accumulation of epicardial adipose tissue (EAT) is considered to be a cardiovascular risk factor independent from visceral adiposity, obesity, hypertension and diabetes. We explored the parameters related to EAT accumulation, aiming to clarify the novel pathophysiological roles of EAT in subjects with type 2 diabetes (T2DM). METHODS: We examined the laboratory values, including cystatinC, and surrogate markers used for evaluating atherosclerosis. EAT was measured as the sum of the adipose tissue area, obtained by plain computed tomography scans in 208 subjects with T2DM but no history of coronary artery disease. RESULTS: EAT correlated positively with age, body mass index (BMI), visceral fat area, leptin, cystatin C and C-peptide, while correlating negatively with adiponectin, estimated glomerular filteration rate (eGFR) and the liver-to-spleen ratio. Multiple linear regression analysis revealed serum cystatin C (ß = 0.175), leptin (ß = 0.536), BMI (ß = 0.393) and age (ß = 0.269) to be the only parameters showing independent statistically significant associations with EAT. When cystatin C was replaced with eGFR, eGFR showed no significant correlation with EAT. In reverse analysis, serum cystatin C was significantly associated with EAT after adjustment in multivariate analysis. DISCUSSION: EAT accumulation and elevated cystatin C have been independently regarded as risk factors influencing atherosclerosis. The strong association between EAT and cystatin C demonstrated herein indicates that EAT accumulation may play an important role in Cystatin C secretion, possibly contributing to cardiometabolic risk in T2DM patients.