The humunosuppressant rapamycin (RAP) has been demonstrated to specifically inhibit the activity of p70 S6 kinase (p700) and subsequent phosphorylation of ribosomal S6 [3HJThymidine and 3H-Labeled Ami Acid ('H-Amino Acid) Incorporation. DNA synthesis was evaluated by incorporation of [3H]thymidine into cells as described (3). General protein synthesis was evaluated by incorporation of 3H-amino acid mixture (Amersham) into cells. After labeling cells with 5 KCi (1 Ci = 37 GBq) ofthe 3H-amino acid mixture per ml, cells were washed twice with ice-cold phosphatebuffered saline (PBS) and then lysed in PBS containing 1% SDS followed by addition of 7% trichloroacetic acid/1% pyrophosphate. The precipitates were loaded on GF/A filters and washed extensively with 7% trichloroacetic acid/1% pyrophosphate. Radioactivity was measured by scintillation counting.Kinase Assay of p70W. Specific activity of p706k was determined by 32p incorporation into S6 peptide in the immune complex as described (4).[ Abbreviations: RAP, rapamycin; p7086k, p70 S6 kinase; eEF-2, elongation factor 2; eEF-la, elongation factor la; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNP, mRNA-ribonucleoprotein complex; PCNA, proliferating cell nuclear antigen; DHFR, dihydrofolate reductase. tTo whom reprint requests should be addressed. 11477The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Embryonic stem (ES) cells have a potential to differentiate into various progenitor cells.Here we investigated the differentiation capacity of mouse ES cells into hepatocytes both in vitro and in vivo. During the culture of embryoid bodies (EBs) derived from ES cells, albumin (ALB) messenger RNA (mRNA) was expressed within 12 days after removal of leukemia inhibitory factor, and ␣-fetoprotein (AFP) mRNA was observed within 9 days without additional exogenous growth factors. In ES cells and early EBs, by contrast, neither ALB mRNA nor AFP mRNA was observed. ALB protein was first detected at day 15 and the level increased with the culture period. The differentiation of EBs facilitated the synthesis of urea with the culture period, whereas early EBs and ES cells produced no urea. These results suggest that cultured EBs contain hepatocytes capable of producing ALB and urea. ES cells and the isolated cells from EBs were transplanted through portal vein to the liver after 30% partial hepatectomy of female mice pretreated with 2-acetylaminofluorene. Four weeks after transplantation with isolated cells from day-9 EBs, ES-derived cells containing Y-chromosome in the liver were positive for ALB (0.2% of total liver cells), whereas teratoma was found in mice transplanted with ES cells or EBs up to day 6. The incidence of teratoma was decreased with the culture duration and no teratoma was observed in the liver transplanted with isolated cells from day-9 EBs. In conclusion, our in vitro and in vivo experiments revealed that cultured EBs contain functional hepatocytes or hepatocyte-like cells. (HEPATOLOGY 2002;36:22-29.)

Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation because of the immaturity of newborn cells compared with adult cells. In contrast to their hematopoietic and mesenchymal potential, it remains unclear whether UCB cells have endodermal competence. Here, with a view to utilize UCB cells for cell transplantation into injured liver, we investigated the hepatic potential of UCB cells both in vitro and in vivo. We determined the most efficient conditions leading UCB cells to produce albumin (ALB). In a novel primary culture system supplemented with a combination of growth/differentiation factors, about 50% of UCB cells in 21-day cultures expressed ALB, and the ALB + cells coexpressed hepatocyte lineage markers. The ALB-expressing cells were able to proliferate in the culture system. Moreover, in the cell-transplantation model into liver-injured severe combined immunodeficient mice, inoculated UCB cells developed into functional hepatocytes in the liver, which released human ALB into the sera of the recipient mice. In conclusion, this study demonstrates that human UCB is a source of transplantable hepatic progenitor cells. Our findings may have relevance to clinical application of UCB-derived cell transplantation as a novel therapeutic option for liver failure.

No abstract

Broiler chicks were reared on either wet litter or dry litter to compare the development of footpad dermatitis (FPD). Broilers reared on wet litter first developed FPD at 14 days of age. Their FPD scores increased sharply after 21 days of age, reaching 2.92 at 42 days. In broilers reared on dry litter, FPD was first observed at 28 days of age, and the FPD score was only 0.70 at 42 days. When 21- or 28-day-old broilers that had been reared on wet litter and had developed FPD were moved to dry litter, the progression of FPD was suppressed or delayed. These results suggest that reducing litter moisture is effective in preventing FPD and suppressing disease progression.