Abstract
The molecular mechanism underlying the initiation of somatic cell reprogramming into induced pluripotent stem cells (iPSCs) has not been well described. Thus, we generated single-cell-derived clones by using a combination of drug-inducible vectors encoding transcription factors (Oct4, Sox2, Klf4 and Myc) and a single-cell expansion strategy. This system achieved a high reprogramming efficiency after metabolic and epigenetic remodeling. Functional analyses of the cloned cells revealed that extracellular signal-regulated kinase (ERK) signaling was downregulated at an early stage of reprogramming and that its inhibition was a driving force for iPSC formation. Among the reprogramming factors, Myc predominantly induced ERK suppression. ERK inhibition upregulated the conversion of somatic cells into iPSCs through concomitant suppression of serum response factor (SRF). Conversely, SRF activation suppressed the reprogramming induced by ERK inhibition and negatively regulated embryonic pluripotency by inducing differentiation via upregulation of immediate early genes, such as c-Jun, c-Fos and EGR1. These data reveal that suppression of the ERK-SRF axis is an initial molecular event that facilitates iPSC formation and may be a useful surrogate marker for cellular reprogramming.
Highlights
The reprogramming mechanisms by which somatic cells acquire embryonic stem cell (ESC) properties, such as self-renewal and pluripotency, have been intensively studied at the molecular level.[1,2,3,4,5] Among these, elucidation of the molecular mechanisms underlying the early steps of reprogramming may provide insight into this phenomenon, which is the reverse of the steps that occur during development, and help to identify other means by which to acquire self-renewal and pluripotency
Generation of reprogramming clones from MEFs To generate reprogramming cell lines, we immortalized mouse embryonic fibroblasts (MEFs) with a reprogramming reporter system, namely, green fluorescent protein (GFP) expression driven by the ESC-specific promoter Oct[4] (Oct4-GFP), which became growth-arrested within several passages in culture
GC5 and JC5 cells exhibited a mesenchymal-toepithelial transition (MET) phenotype within 3 days of culture in MEF medium containing Dox (Supplementary Figure 2a), developed an ESC-like morphology and expressed GFP when cultured in knockout serum replacement (KSR) medium containing leukemia inhibitory factor (LIF) and Dox (KSR/LIF/Dox; Figure 2c)
Summary
The reprogramming mechanisms by which somatic cells acquire embryonic stem cell (ESC) properties, such as self-renewal and pluripotency, have been intensively studied at the molecular level.[1,2,3,4,5] Among these, elucidation of the molecular mechanisms underlying the early steps of reprogramming may provide insight into this phenomenon, which is the reverse of the steps that occur during development, and help to identify other means by which to acquire self-renewal and pluripotency. The representative characteristics of the early stage of reprogramming are rapid proliferation[6] and mesenchymal-toepithelial transition (MET),[7,8] the roles of these phenotypic signatures of reprogramming are controversial.[9,10] Gene expression profiles of the reprogramming process have identified several genes, such as Esrrb, Utf[1] and EpCAM, that mark the initial phase.[11,12] expression of these genes is variable during the initial stage;[11,12] it is unclear whether they can predict successful reprogramming
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