In addition to their contribution to basic research such as stem cell biology and early Rapamycin CAS human development, hES cells have great potential as source of cells for therapeutic uses. In order to reduce the risks of cross transfer of pathogens from xenogeneic feeder or conditioned medium, an autogeneic feeder cell system, comprising fibroblast like cells differentiated from hES cells, was developed to grow undifferentiated and pluri potent hES cells for their medical applications. A fee der free culture using medium conditioned by autogeneic feeder cells is desirable in order to use hES cells as tools for drug development and toxicity testing. In our laboratory, five hES cell lines had been derived, and one line hES T3 with normal female karyotype was used to establish autogeneic feeder cells with capa city to support the growth of undifferentiated hES cells.
In this investigation, a feeder free culture on Matrigel in medium conditioned by these autogeneic feeder cells was established to maintain the undifferentiated growth of hES cells, and the gene expression profiles of mRNAs, microRNAs and proteins were further shown to be very similar between the undifferentiated hES cells grown on autogeneic feeder and its conditioned medium, as well as MEF feeder and MEF conditioned medium. Methods Undifferentiated growth of hES cells on MEF feeder and MEF conditioned medium Human embryonic stem cell line hES T3, which is one of the five hES cell lines derived in our laboratory with insti tutional review board approval and informed consent by couples undergoing IFV treatment in Taiwan, exhibits normal female karyotype, and it has been con tinuously cultured on mitomycin C mitotically inactivated MEF feeder in hES medium under 5% CO2 at 37 C and underwent freezing thawing processes.
The hES culture medium consisted of DMEM F12 supplemented with 20% KSR, 1% non essential amino acids, 1 mM L glutamine, 0. 1 mM b mercaptoethanol, and 4 ng ml human basic fibroblast growth factor. Routine pas sages of hES T3 cells every 5 7 days were done with collagenase treatment and mechanical scrape. The cryopreserved stock of hES T3 cells were continuously maintained on MEF feeder for additional 14 passages, and these the hES T3 cells were designated as T3 MEF. The MEF cells were cultured in MEF medium overnight, and the mitotically inactivated MEF cells were maintained in hES medium containing 4 ng ml bFGF.
After 24 h, the MEF conditioned medium was collected and filtered through 0. 2 um membrane as previously described. The culture dish was coated with Matrigel diluted with DMEM F12 overnight at 4 C. The cryopreserved stock of hES T3 cells were continuously maintained on feeder free Matrigel coated Dacomitinib dish in MEF conditioned medium for 12 passages, and these hES T3 cells were designated as T3 CMMEF.