ABSTRACT
Pluripotency can be induced in somatic cells by overexpressing transcription factors, including POU class 5 homeobox 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and myelocytomatosis oncogene (c-MYC). However, some induced pluripotent stem cells (iPSCs) exhibit defective differentiation and inappropriate maintenance of pluripotency features. Here we show that dynamic regulation of human endogenous retroviruses (HERVs) is important in the reprogramming process toward iPSCs, and in re-establishment of differentiation potential. During reprogramming, OCT3/4, SOX2, and KLF4 transiently hyperactivated LTR7s--the long-terminal repeats of HERV type-H (HERV-H)--to levels much higher than in embryonic stem cells by direct occupation of LTR7 sites genome-wide. Knocking down LTR7s or long intergenic non-protein coding RNA, regulator of reprogramming (lincRNA-RoR), a HERV-H-driven long noncoding RNA, early in reprogramming markedly reduced the efficiency of iPSC generation. KLF4 and LTR7 expression decreased to levels comparable with embryonic stem cells once reprogramming was complete, but failure to resuppress KLF4 and LTR7s resulted in defective differentiation. We also observed defective differentiation and LTR7 activation when iPSCs had forced expression of KLF4. However, when aberrantly expressed KLF4 or LTR7s were suppressed in defective iPSCs, normal differentiation was restored. Thus, a major mechanism by which OCT3/4, SOX2, and KLF4 promote human iPSC generation and reestablish potential for differentiation is by dynamically regulating HERV-H LTR7s.
Subject(s)
Endogenous Retroviruses/genetics , Endogenous Retroviruses/physiology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/virology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cellular Reprogramming/genetics , Cellular Reprogramming/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Embryonic Stem Cells/virology , Epigenesis, Genetic , Gene Expression , Gene Knockdown Techniques , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Induced Pluripotent Stem Cells/virology , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/physiology , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/physiology , Pluripotent Stem Cells/physiology , RNA, Long Noncoding/antagonists & inhibitors , RNA, Long Noncoding/genetics , RNA, Viral/antagonists & inhibitors , RNA, Viral/genetics , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/physiologyABSTRACT
We examined the gene expression and DNA methylation of 49 human induced pluripotent stem cells (hiPSCs) and 10 human embryonic stem cells and found overlapped variations in gene expression and DNA methylation in the two types of human pluripotent stem cell lines. Comparisons of the in vitro neural differentiation of 40 hiPSCs and 10 human embryonic stem cells showed that seven hiPSC clones retained a significant number of undifferentiated cells even after neural differentiation culture and formed teratoma when transplanted into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression levels of several genes, including those expressed from long terminal repeats of specific human endogenous retroviruses. These data demonstrated a subset of hiPSC lines that have aberrant gene expression and defective potential in neural differentiation, which need to be identified and eliminated before applications in regenerative medicine.
