Protein-based reprogramming is not by the contamination of protein-donor ES cell. To rule out the possibility of ES cell contamination during the preparation of ES-derived extract proteins, further iPS cell generation was performed using adult somatic cells of a different genetic strain from protein-donor ES cells. The donor of ES-derived extract proteins was a C57 background, and the recipient of ES proteins (a target cell of reprogramming) was a FVB background. (A) Bright-field morphology of ES-like colonies developed from FVB background skin fibroblasts (sFB) using C57 ES cell–derived extract proteins. (B) Multiple microsatellite markers were applied to confirm the origin of the protein-iPS cells. PCR was performed using genomic DNA from each cell type. (C) To further verify the cellular origin, the sizes of amplified PCR products were validated using fluorescent 5′-FAM or 5′-HEX primers. The Genescan 500ROXTM size standard served as an internal control. (D) The chromosomes of ES cell, adult fibroblast, and protein-iPS cell were diploid and normal karyotype.