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.

Telomerase activity was analysed in 100 neuroblastoma cases. Although telomerase activity was not detected in normal adrenal tissues or benign ganglioneuromas, almost all neuroblastomas (94%) did express it, suggesting an important role for telomerase in neuroblastoma development. Neuroblastomas with high telomerase activity had other genetic changes (for example, N-myc amplification) and an unfavourable prognosis, whereas tumours with low telomerase activity were devoid of such genetic alterations and were associated with a favourable prognosis. Three neuroblastomas lacking telomerase activity regressed (stage IVS). Thus telomerase expression may be required as a critical step in the multigenetic process of tumorigenesis, and two different pathways may exist for the development of neuroblastoma.

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Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation or liver-directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth-Holm-Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth-Holm-Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naïve liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naïve liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders. (HEPATOLOGY 2005; 41:132-140.)

We examined human telomerase reverse transcriptase (hTERT) protein distribution by immunohistochemistry in cultured cells and tissue sections. Cells with telomerase activity had nuclear positive signals whereas cells without telomerase activity did not. In most normal epithelial tissues, hTERT expression was prominent in the early proliferative descendent progenitors cells. In cancers with high telomerase activity, hTERT expression was detected in almost all neoplastic cells and correlated with telomerase activity levels, whereas cancers with low telomerase activity had fewer hTERT-positive cancer cells. In pediatric neuroblastomas with a favorable outcome, both the percentage of positive cells and the signal intensities of each hTERT-expressing cell decreased. These studies indicate that detection of telomerase at the cellular level is achievable and may have utility in cancer diagnostics.