ABSTRACT
A reprogrammable transgenic mouse strain, called Col1a1 4F2A-Oct4-GFP, was bred for the present study. Because the somatic cells of this mouse strain contain only two copies of each Yamanaka factor, these animals are inefficient at producing induced pluripotent stem cells (iPSCs; approx. 0.005%) under traditional culture conditions. With an optimized culture condition, the iPSC production rate of mouse embryonic fibroblasts (MEFs) of Col1a1 4F2A-Oct4-GFP mice (MEFCol1a14F2A-Oct4-GFP ) was increased to approximately 8%. Further, promotion of cell proliferation by serum supplementation did not enhance iPSC production. Inhibition of transforming growth factor ß (TGF-ß) in the serum by SB431542 neither affected the growth rate of MEFCol1a14F2A-Oct4-GFP nor promoted iPSC production. However, the use of the gamma-irradiated STO-NEO-LIF (γSNL) cells to serve as feeders for iPSC production resulted in a 5-fold higher rate of iPSC production than the use of γMEFICR feeders. Interestingly, the use of SB431542 with the γMEFICR -adopted system could eliminate this difference. RT-PCR-based comparative analysis further demonstrated that TGF-ß expression was 10-fold higher in γMEFICR than in γSNL cells. Consistent with previous reports, mesenchymal to epithelial transition was found to participate in the initial steps of reprogramming in the specific context of MEFCol1a14F2A-Oct4-GFP . Moreover, we found that the initial seeding density is one of the pivotal factors for determining a high efficiency of iPSC generation. The iPSCs efficiently generated from our MEFCol1a14F2A-Oct4-GFP resembled mouse embryonic stem cells (mESCs) in aspects of teratoma formation and germline transmission. Depending on the culture conditions, our Col1a1 4F2A-Oct4-GFP mouse system can generate bona fide iPSCs with variable efficiencies, which can serve as a tool for interrogating the route taken by cells during somatic reprogramming.
Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells/drug effects , Transforming Growth Factor beta/physiology , Animals , Cell Division/drug effects , Cells, Cultured , Coculture Techniques , Collagen Type I/genetics , Collagen Type I, alpha 1 Chain , Culture Media/pharmacology , Doxycycline/pharmacology , Fibroblasts/physiology , Fibroblasts/radiation effects , Gamma Rays , Gene Expression Regulation/drug effects , Induced Pluripotent Stem Cells/cytology , Mice , Mice, Knockout , Recombinant Fusion Proteins/metabolism , Teratoma/pathology , Transcription Factors/pharmacology , Transforming Growth Factor beta/pharmacology , TransgenesABSTRACT
OBJECTIVE: To investigate the expression of human epidermal growth factor receptor 2 (HER-2/neu) in hepatocellular carcinoma (HCC) patients and its clinical significance. METHODS: The expressions of HER-2/neu were detected by SP immunohistochemistry method in 30 patients with HCC, 10 with portal cirrhosis of the liver and 10 with normal liver. RESULTS: The positivity rate of HER-2/neu was markedly higher in HCC patients than in those with portal cirrhosis and normal liver (Chi(2)=6.482, P=0.032). The expression of HER-2/neu was closely correlated to portal cirrhosis of the liver (P=0.041), tumor invasion (P=0.028) and Edmondson grades (P=0.012). The average survival time was significant shorter in patients with HER-2/neu-positive tumor than in those with HER-2/neu-negative tumor (P=0.036). CONCLUSION: The expression of HER-2/neu may play a role in the invasion, metastasis and progression of HCC. The patients positive for HER-2/neu in the HCC tissues have generally poor prognosis.
