To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1×10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well micro-plates at about 5×10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.

Side-population (SP) cells have been shown to be highly enriched stem cells. We investigated whether an immortalized, nontumorigenic human liver cell line, THLE-5b, contains SP cells. Flow cytometry analysis after Hoechst 33342 staining demonstrated that the THLE-5b line contained a small component of SP cells. These SP cells were essentially eliminated by treatment with verap-amil and expressed higher levels of ABCG2 mRNA than non-SP cells. In addition, the level of these SP cells detected by Hoechst 33342 staining was affected by the experimental conditions including the incubation medium. This is the first report of the presence of SP cells in the immortalized, nontumorigenic human liver cell line.

ES mice that are derived completely from embryonic stem (ES) cells can be obtained by tetraploid embryo complementation. Many neonate ES mice die because of respiratory distress, but it is not clear what contributes to the phenomenon. Using five microsatellite DNA markers, we confirmed that our ES mice were completely derived from ES cells and contained no tetraploid component. The neonatal ES mice that exhibited respiratory distress were tested for surfactant protein B (SP-B) expression by Western blotting. These mice had no SP-B expression, and even apparently healthy adult ES mice had decreased SP-B levels and aberrant SP-B phenotypes. These data suggest that the expression of SP-B protein is an important factor in the survival of ES mice to term and adulthood.

We studied the expression and mutation of thyroid transcription factor-1 (TTF-1) gene in 92 cases of lung carcinomas comprised of lung adenocarcinoma (36 cases), squamous cell lung carcinoma (42 cases), small cell lung carcinoma (8 cases), and large cell lung carcinoma (6 cases) to investigate whether TTF-1 gene mutation predisposed to the development of lung cancer. Normal lung tissues were obtained from each of the 92 patients. The tissues served as controls. Polymerase chain reaction–single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and DNA sequencing were used to analyze TTF-1 gene mutation and its relationship with the carcinogenesis of lung cancer. We detected the expression of TTF-1 protein and messenger RNA (mRNA) in paraffin-embedded lung carcinomas and their normal lung tissues by tissue microarray. TTF-1 protein and mRNA intensities were measured by Leica-Q500 MC Image Analysis System to reveal their correlation with TTF-1 mutation in lung carcinomas. TTF-1 gene missense and synonymous mutation are present in lung carcinomas with the mutation rate of 16%. TTF-1 protein and mRNA are higher in normal lung tissues than in different lung carcinomas. TTF-1 gene mutation is correlated with the loss of TTF-1 protein and mRNA. The analyses of TTF-1 gene missense mutation and synonymous mutation and the loss of TTF-1 protein and mRNA can be regarded as the important indexes of molecular pathology of lung carcinoma.

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H ,K -adenosine triphosphatase (H ,K -ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.

Bone marrow stromal cells (MSCs) have the capability of differentiating into mesenchymal and non-mesenchymal lineages. In this study, MSCs isolated from adult Sprague-Dawley rats were cultured to proliferation, followed by in vitro induction under specific conditions. The results demonstrated that MSCs were transdifferentiated into cells with the Schwann cell (SC) phenotypes according to their morphology and immunoreactivities to SC surface markers including S-100, glial fibrillary acidic protein (GFAP) and low-affinity nerve growth factor receptor (p75). Consequently, rat adult MSCs can be induced in vitro to differentiate into SC-like cells, thus developing an abundant and accessible SC reservoir to meet the requirements of constructing tissue engineered nerve grafts for peripheral nerve repair.

TGF-β1 is thought to decrease the proportion of embryonic pancreatic β-cells with respect to α-cells, whereas glucose is thought to enhance β-cell proportions in rats. However, chick pancreatic cells may respond in a dissimilar way to glucose. Thus, the effect of TGF-β1 on the proportion of β-cells in embryonic chick dorsal pancreatic buds (DPBs) in vitro was tested with short-term exposure to high levels of glucose. Five-day-old chick DPBs were cultured on growth factor-reduced Matrigel, which contains reduced levels of growth factors including TGF-β1, and a variety of culture media with and without high levels of glucose. TGF-β1 reduced the proportion of β-cells, as expected. A similar decrease in the proportion of β-cells occurred in the presence of high levels of glucose.