Residual Expression of Reprogramming Factors Affects the Transcriptional Program and Epigenetic Signatures of Induced Pluripotent Stem Cells

Abstract

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 Gtl2ON vs Gtl2OFF iPSC clones. Our findings provide new insights into the reprogramming process and emphasize the importance of generating iPSCs free of any residual transgene expression.

Sox9 Reprogrammed Dermal Fibroblasts Undergo Hypertrophic Differentiation In Vitro and Trigger Endochondral Ossification In Vivo

“…Indeed, similar to our findings, the choice of the promoter system for driving transgene expression is known to affect the transcriptional program and epigenetic signature in iPSCs (Sommer et al, 2012). iPSC clones that lost their transgene expression, were remarkably more similar to their native counterparts (ESCs), whereas others that contained a continuous expression of transgenes prevented them from acquiring the native transcriptional profile (Sommer et al, 2012).…”

Induction of Hepatocyte Differentiation in Human Pluripotent Stem Cells

“…Indeed, differentiation potential depends on the hiPS cells used [102] , and there are differences between hiPS cell populations in the efficiency of differentiation to hepatocytes, as evidenced by differences in albumin expression levels [103] . The reprogramming factors themselves affect the differentiation of iPS cells to specific cell fates [104] . Although the results originate from mouse iPS cells, there are differences in the methylation sites between iPS cells reprogrammed with different factors.…”

Relevance of the plasma membrane calcium-ATPase in the homeostasis of calcium in the fetal liver

“…37,40,41 In other studies where reprogramming is performed in the absence of transgenes, no transcriptomic differences were identified in cells that were reprogrammed with or without c-myc. 42 Of particular interest is the finding that among more than 2,000 genes that exhibited significant differential expression when the transgene is absent (most of them related to epigenetic regulation and chromatin assembly), the housekeeping PMCA isoforms were expressed. Furthermore, PMCA4 appeared to be overexpressed, while PMCA1 was underexpressed.…”

“…Furthermore, PMCA4 appeared to be overexpressed, while PMCA1 was underexpressed. 42 Although the meaning of these results is not readily apparent, they may be understood in terms of the unique features of each isoform associated to the specific demands that the microenvironment might impose.…”