Objective:

To generate human fibroblast feeder cells able of supporting human induced pluripotent stem cells (hiPSCs), through the introduction of genes that enhance cell survival and growth.

Methods:

Lentiviral particles were produced in HEK 293T cells using vectors encoding human KRAS, FGF2, and hTERT. Viral titers were quantified by p24. Human fibroblast cells were transduced with these three lentiviral vectors and subsequently selected with puromycin to enrich for cells expressing the transgenes. The selected fibroblasts were then γ-irradiated to inhibit proliferation, enabling their use as feeder cells.

Results:

Lentiviral production was successfully, and viral titers were sufficient for efficient transduction. After selection, a stable population of transgene-expressing fibroblasts was obtained. Irradiated fibroblasts maintained their morphology and adherence properties, making them suitable for use as a feeder layer.

Conclusion:

We successfully generated a stable, irradiated human fibroblast feeder cell line through the expression of KRAS, FGF2, and hTERT, which is now being evaluated for its capacity to support hiPSC reprogramming derivation, clonal expansion and genome engineering using CRISPR Cas9 tools