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).

Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets.

Hepatic tissue engineering using primary hepatocytes has been considered a valuable new therapeutic modality for several classes of liver diseases. Recent progress in the development of clinically feasible liver tissue engineering approaches, however, has been hampered mainly by insufficient cell-to-cell contact of the engrafted hepatocytes. We developed a method to engineer a uniformly continuous sheet of hepatic tissue using isolated primary hepatocytes cultured on temperature-responsive surfaces. Sheets of hepatic tissue transplanted into the subcutaneous space resulted in efficient engraftment to the surrounding cells, with the formation of two-dimensional hepatic tissues that stably persisted for longer than 200 d. The engineered hepatic tissues also showed several characteristics of liver-specific functionality. Additionally, when the hepatic tissue sheets were layered in vivo, three-dimensional miniature liver systems having persistent survivability could be also engineered. This technology for liver tissue engineering is simple, minimally invasive and free of potentially immunogenic biodegradable scaffolds.

Establishment and characterization of a rat lung adenocarcinoma cell line with low malignant potential.

To investigate characteristic of lung adenocarcinoma growth behavior, we have established a cell line (rat lung cancer in Nara (RLCNR)) from a tumor induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in a rat. This clone shows an epithelial cell morphology and grows as sheets in culture with an approximate doubling time of 19.2 h. Ultrastructurally, the RLCNR contains lamellar bodies in cytoplasm and the microvilli are present at the free cell surfaces. The line features well-developed desmosomes. The modal chromosome number of 42 is the same as for normal rat cells and its frequency was established to be 80.5%. To evaluate tumorigenicity, appropriate numbers of the cells were transplanted into syngeneic rats, but no tumor formation occurred. Genetic analyses revealed the RLCNR to have a GGT to GAT mutation at codon 12 of Ki-ras, but no p53 alteration. p16 gene expression was lost, associated with hypermethylation of CpG sites in the 5' upstream region of the gene. These results indicate that the present newly established cell line originated from an alveolar type II lesion of the lung. It should be useful for further investigation of in vivo growth mechanisms, especially tumor progression, of lung adenocarcinomas, since it has low malignant potential.