Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating 
viral vectors has been widely employed to generate induced pluripotent stem cell 
(iPSC) lines from both normal and disease-specific somatic tissues, providing an 
invaluable resource for medical research and drug development. Residual 
reprogramming transgene expression from integrated viruses nevertheless alters 
the biological properties of iPSCs and has been associated with a reduced 
developmental competence both in vivo and in vitro. We performed transcriptional 
profiling of mouse iPSC lines before and after excision of a polycistronic 
lentiviral reprogramming vector to systematically define the overall impact of 
persistent transgene expression on the molecular features of iPSCs. We 
demonstrate that residual expression of the Yamanaka factors prevents iPSCs from 
acquiring the transcriptional program exhibited by embryonic stem cells (ESCs) 
and that the expression profiles of iPSCs generated with and without c-Myc are 
indistinguishable. After vector excision, we find 36% of iPSC clones show normal 
methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent 
iPSC lines. Furthermore, we show that the reprogramming factor Klf4 binds to the 
promoter region of Gtl2. Regardless of Gtl2 methylation status, we find similar 
endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2(ON) 
vs Gtl2(OFF) iPSC clones. Our findings provide new insights into the 
reprogramming process and emphasize the importance of generating iPSCs free of 
any residual transgene expression.