(A) Use of a ubiquitously and conditionally expressed genetic marker to exclude that iPS cells acquire hepatocyte function by fusion with blastocyst-derived hepatocytes. The FAH-deficient blastocysts used for iPS cell injection were also heterozygous for the Rosa26 reporter (R26R; ref. 32). Therefore, all cells derived from the blastocyst activate R26R (X-gal staining, blue) in response to expression of Cre recombinase. Only cells that developed by direct differentiation of the iPS cells remain unlabeled in chimeric mice. Hepatocytes isolated at approximately 95% purity from a chimeric mouse with approximately 90% liver repopulation (left) and a R26R heterozygous control mouse (right) were infected with a Cre-expressing adenovirus (Supplemental Figure 7). Activation of R26R in all hepatocytes from the R26R heterozygous mouse, but in less than 10% of the hepatocytes isolated from the chimeric mouse, shows that iPS cell–derived hepatocytes lack R26R and emerged independently of cell fusion. Scale bars: 50 μm. (B) Fusion-independent iPS cell differentiation into hepatocytes was confirmed by semiquantitative PCR analysis of DNA from the isolated hepatocytes for markers specific for the iPS cells (homozygous wild-type for Fah, homozygous negative for R26R and positive for GFP) versus markers specific for the blastocysts (homozygous knockout for Fah, heterozygous for R26R and negative for GFP). Due to the high level of liver repopulation in this chimeric mouse, isolated hepatocytes were predominantly iPS cell derived. A substantial fraction of the cells in the liver are not hepatocytes and are thus not replaced in response to FAH deficiency. As expected, blastocyst contribution to these cell types was maintained in the whole liver sample after NTBC withdrawal.