Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells
Figure 5
Viable mice derived from gene-corrected FAHgc-iPS cells via tetraploid embryo complementation.
(A) A representative litter of FAHgc-IPS cell–derived neonatal pups. One of the pups failed to initiative breathing (*) after caesarean section delivery on E19.0. (B) Immunohistological staining for FAH demonstrated that about 20% of the hepatocytes in a newborn's liver expressed the FAH protein. (C) FAH−/− liver tissue without gene correction was used as a negative control, confirming the specificity of the antibody staining. (D) Wild-type liver was used as a positive control to confirm the sensitivity of the FAH immunohistochemistry. All hepatocytes, but no endothelial or blood cells, stained positive for FAH. (E) Two healthy #7.2 (SFFV promoter) FAHgc-iPS cell–derived mice under supplementation with NTBC. (F and G) FAH staining on the liver of these mice in (E) indicated FAH expression in about 25% of the liver cells. (H) Immunohistochemistry counterstaining for EGFP demonstrated that all FAH-positive cells in panel (G) also stained positive for EGFP. (I) FAH staining in #7.2 (SFFV promoter) FAHgc-iPS cell–derived mouse liver 5 wk after NTBC withdrawal showed FAH expression in most of the liver cells. (J) FAH staining in #7.8 (TTR promoter) FAHgc-iPS cell–derived mouse liver indicates FAH expression in about 15% of the liver cells. (K) Immunohistochemistry counterstaining for EGFP demonstrates that all FAH-positive cells in panel J also stained positive for EGFP. (L) FAH staining in #7.8 (TTR promoter) FAHgc-iPS cell–derived mouse liver 4 wk after NTBC withdrawal indicates FAH expression in most of the liver cells. (M) Absence of FAH-positive staining in a kidney sample of iPS line #7.8–derived mice confirmed the tissue specificity of the hepatic TTR promoter. (N) Estimated gene copy numbers of the FAH-P2a-EGFP construct in both FAHgc-iPS cell lines #7.1 and #7.2 as well as in liver tissue of an FAHgc-iPS cell #7.2–derived mouse. (O) FAH mRNA expression levels relative to adult liver tissue (set as 100%) in FAHgc-iPS cell lines and in liver samples of an FAHgc-iPS cell–derived mouse (“on” and “off” NTBC). Scale bars, 100 µm.