(A) Relative postnatal weight gain in chimeric mice compared with wild-type and FAH-deficient (KO) controls. Chimeric mice were separated into 2 groups, based on high versus no/low contribution of iPS cells to genomic DNA from digits (Supplemental Figure 1). NTBC withdrawal was initiated on P6. In contrast to KO controls and mice with no/low levels of digital chimerism, mice with high levels of digital chimerism did not require reinstatement of NTBC treatment after P22 for growth and survival. All control mice were on the 129S4 strain background, while both iPS cells and blastocysts were on a mixed 129S4 and C57BL/6 background. C57BL/6 neonates grow faster, which explains the slight difference in weight gain between control and chimeric mice between P6 and P19. (B) Quantitative RT-PCR shows that growth and survival in the absence of NTBC are strictly correlated with Fah expression in the liver. Liver Fah expression is detectable in all mice with a high iPS cell contribution to digits but not in mice with no/low levels of digital chimerism. Fah expression approaches wild-type levels with time off NTBC, increasing from approximately 50% (range, 33%–69%) in mice analyzed at P28 (left red bar) to approximately 80% (range, 76%–78%) in mice analyzed at P70 (right red bar). Maximum Fah expression levels in iPS cell chimeric mice are similar to levels found after approximately 100% liver repopulation in mice generated by injection of normal ES cells into FAH-deficient blastocysts (ESCs). (C–F) FAH immunostaining (red). Representative liver sections show that mice with (C) no or (D) low digital chimerism lack FAH-expressing cells. Livers of mice with high levels of digital chimerism show repopulation with FAH-positive cells, between approximately (E) 50% at P28 and (F) 100% at P70. Nuclei are stained blue. Scale bars: 100 μm. Data represent mean ± SEM. *P < 0.05; **P < 0.005; ^#P > 0.05.