hES T3 cells were transferred into feeder free and noncoated plate in DMEM supplemen ted with 10% FBS under 5% CO2 at 37 C. After selleckchem Pacritinib 10 days, cells appeared as fibroblast like morphology, that is, flat cells with elongated nucleus and branching pseudopodia. These hES T3 differentiated fibroblast like cells are designated as T3HDF. The expression of tran scription factors OCT4, SOX2 and NANOG, which were highly expressed in T3 MEF cells, was shown to be down regulated in differentiated T3HDF cells. The expression profiles of mRNAs and miRNAs between T3 MEF and T3HDF cells were also found to very different. These T3HDF cells were passaged using trypsin every 4 days or cryopreserved.
Undifferentiated growth of hES cells on T3HDF feeder and T3HDF conditioned medium The differentiated fibroblast like T3HDF cells were inactivated using mitomycin C and used as autogeneic feeder layer in hES medium to main tain the continuously undifferentiated growth of hES T3 cells for additional 14 passages. These hES T3 cells grown on T3HDF feeder were designated as T3 HDF. The T3HDF cells were cultured in DMEM medium overnight, and the mitotically inactivated T3HDF were maintained in hES medium containing 4 ng ml bFGF. After 24 h, the T3HDF conditioned medium was col lected and filtered through 0. 2 um membrane. The culture dish was coated with Matrigel diluted with DMEM F12 overnight at 4 C. The hES T3 cells were first grown on T3HDF feeder for 4 passages and then on Matrigel in T3HDF conditioned medium for additional 4 passages.
The hES T3 cells grown on feeder free Matrigel coated dish in T3HDF conditioned medium were designated as T3 CMHDF Staining of OCT4 and NANOG T3 HDF and T3 CMHDF, as well as T3 MEF and T3 CMMEF, colonies were fixed by 4% paraformaldehyde and permeabilized using 0. 5% Triton X 100 in the culture dishes. The immunostaining with rabbit polyclonal anti bodies against human OCT4 and NANOG were detected with goat anti rabbit IgG as described previously. Extraction of total RNAs Total RNAs from approximately 1 �� 106 cells of T3 HDF and T3 CMHDF on 10 cm plate were extracted using TRIZOL reagent, and the same total RNAs from each sample were used for both mRNA microarray ana lysis and miRNA quantification. The mRNA profilings of T3 HDF and T3 CMHDF cells were analyzed using Affymetrix Human Genome U133 plus 2.
0 GeneChip according to the Manufacturers proto cols by the Microarray Core Facility of National Research Pro gram for Genomic Medicine of National Science Council in Taiwan as previously described. This Affymetrix GeneChip contains 54,675 probe sets to analyze the expression levels of 47,400 transcripts and variants, includ ing 38,500 well characterized Brefeldin_A human genes. GeneChips from the hybridization experiments were read by the Affy metrix GeneChip scanner 3000, and raw data were pro cessed using Affymetrix GeneChip Operating Software MAS5.