Abstract
Reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) often generates partially reprogrammed iPSCs (pre-iPSCs), low-grade chimera forming iPSCs (lg-iPSCs) and fully reprogrammed, high-grade chimera production competent iPSCs (hg-iPSCs). Lg-iPSC transcriptome analysis revealed misregulated Dlk1-Dio3 cluster gene expression and subsequently the imprinting defect at the Dlk1-Dio3 locus. Here, we show that germ-cell marker Dppa3 is present only in lg-iPSCs and hg-iPSCs, and that induction with exogenous Dppa3 enhances reprogramming kinetics, generating all hg-iPSCs, similar to vitamin C (Vc). Conversely, Dppa3-null fibroblasts show reprogramming block at pre-iPSCs state and Dlk1-Dio3 imprinting defect. At the molecular level, we show that Dppa3 is associated with Dlk1-Dio3 locus and identify that Dppa3 maintains imprinting by antagonizing Dnmt3a binding. Our results further show molecular parallels between Dppa3 and Vc in Dlk1-Dio3 imprinting maintenance and suggest that early activation of Dppa3 is one of the cascades through which Vc facilitates the generation of fully reprogrammed iPSCs.
Highlights
Reprogramming of mouse somatic cells into induced pluripotent stem cells often generates partially reprogrammed iPSCs, low-grade chimera forming iPSCs and fully reprogrammed, high-grade chimera production competent iPSCs
We evaluate whether these germ cells (GCs) marker genes, in particular Dppa[3], play any role in the maintenance of Dlk1-Dio[3] imprinting during the generation of iPSCs
Advances in understanding the process of somatic cell reprogramming towards iPS cells have proposed three phases in reprogramming: initiation, marking mesenchymal-to-epithelial transition; maturation and stabilization, with pre-iPSCs and characterized by silencing of exogenous reprogramming factors and activation of endogenous pluripotency-related genes, such as Dppa[3], Sox[2] and Dnmt3l7 (Fig. 1a)
Summary
Reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) often generates partially reprogrammed iPSCs (pre-iPSCs), low-grade chimera forming iPSCs (lg-iPSCs) and fully reprogrammed, high-grade chimera production competent iPSCs (hg-iPSCs). Our results further show molecular parallels between Dppa[3] and Vc in Dlk1-Dio[3] imprinting maintenance and suggest that early activation of Dppa[3] is one of the cascades through which Vc facilitates the generation of fully reprogrammed iPSCs. Pluripotent stem cells boast the differentiation potential virtually into any cell type of the body, and hold a great promise for regenerative medicine applications[1]. Short DNA sequences called imprinting control regions (ICRs) are methylated on either the maternal or paternal allele to regulate expression of the imprinted gene in cis[11]. A pool of primordial germ cells (PGCs) gives rise to progenitors of adult gametes After their specification and up on their arrival at the genital ridge, these PGCs undergo demethylation of the whole genome including an erasure of parent-specific methylation marks of imprinted genes, the so-called imprints[14]. Dppa[3] is found to be associated with the intergenic differentially methylated region (IG-DMR) of the Dlk1-Dio[3] and to counteract the binding of Dnmt3a to this region during reprogramming
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