Transgenic human fetal fibroblasts as feeder layer for human embryonic stem cell lineage selection

Sidhu KS, Lie KH, Tuch BE
Source: Stem Cells Dev
Publication Date: (2006)
Issue: 15(5): 741-7
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
Nucleofector® I/II/2b
The Envy hESC line was transfected with green fluorescent protein–small interfering RNA (GFP-siRNA) plasmids (Promega) by using Amaxa Biosystem Nucleofector™ II from Germany, and stably integrated clones were selected using tHFFs (transgenic human fetal fibroblasts) in the presence of puromycin.
Successful gene targeting in human embryonic stem (hES) cells requires the use of primary fibroblast feeder layers, which assist in the maintenance of the pluripotent state of hES cells. Such feeder layers must also survive any further selection strategy for hES cells. Here we report the production of a novel transgenic human fetal fibroblast (tHFF) as a feeder layer that is resistant to puromycin and can be used for gene targeting and selection of positive clones in hES cells. tHFFs survive under a wide range of puromycin concentrations (0.5-2 microg/ml) and also supports the undifferentiated growth of hES cells. We have demonstrated here that tHFFs are suitable for selecting Envy-hES cells that were transfected with a green fluorescent protein-small interfering RNA (GFP-siRNA) plasmid construct to induce GFP gene down-regulation. The later studies were designed to isolate and propagate stably knockdown cells. tHFFs thus can be used for targeting other genes that would serve as a model to select and understand the differentiation process in hES cells.