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.
Highlights
The discovery that differentiated adult cells can be reprogrammed to a state of pluripotency through the introduction of a defined set of transcriptional regulators has opened new avenues for understanding and treating degenerative diseases [1]
The degree of transcriptional dysregulation in transgene-carrying induced pluripotent stem cell (iPSC) appears to be correlated with residual transgene activity and relatively small (1.5- to 2.5-fold) increases in exogenous reprogramming factor expression are sufficient to elicit genome-wide transcriptional changes that can be identified by Principal Components Analysis (PCA)
We provide evidence indicating that i) persistent transgene expression prevents iPSCs from attaining an embryonic stem cells (ESCs)-like transcriptional program; ii) iPSCs reprogrammed with and without c-Myc exhibit highly similar gene-expression profiles; iii) reprogramming results in aberrant imprinting of the Dlk1-Dio3 locus in some but not all iPSC clones generated with our polycistronic cassette; and iv) Klf4 binds strongly to the Gtl2 promoter and shows decrease binding upon removal of the reprogramming transgenes
Summary
The discovery that differentiated adult cells can be reprogrammed to a state of pluripotency through the introduction of a defined set of transcriptional regulators has opened new avenues for understanding and treating degenerative diseases [1]. Residual expression of the integrated viral transgenes in the reprogrammed cells, has been shown to affect their biological properties both in vivo and in vitro [12,13]. In this context, it is important to evaluate the overall gene dysregulation caused by the presence of the transgenes if transgene-carrying iPSCs are to be employed for drug screening, tissue development or disease modeling. The extent to which persistent expression of the reprogramming factors perturbs the transcriptional program of iPSCs has not been systematically assessed It remains unclear whether iPSCs derived with or without c-Myc differ when comprehensively compared by global gene expression profiling
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