Abstract
The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.
Highlights
Induced pluripotent stem cells can be generated by overexpression of core pluripotency factors [1] and represent an important tool for studying development and regulatory mechanisms underlying pluripotency
We have investigated the underlying mechanisms by which the new reversible dual G9a/DNA methyl transferase (DNMT) inhibitor increases the efficiency of conversion to Induced pluripotent stem cells (iPSC), that resulted in a general chromatin relaxation that facilitates the engagement of transcription factors to the genome at refractory binding regions and improves the early events during cell reprogramming of somatic cells to iPSCs
We showed that a new first-in-class dual and reversible G9a/DNMT inhibitor compound [21] improves reprogramming efficiency of human cells inducing heterochromatin relaxation, facilitating the engagement of transcription factors to the genome at refractory binding regions and promoting the early events during cell reprogramming
Summary
Induced pluripotent stem cells (iPSC) can be generated by overexpression of core pluripotency factors [1] and represent an important tool for studying development and regulatory mechanisms underlying pluripotency. Recent studies have enabled large-scale genomic, epigenomic and proteomic profiling of cells as they acquire pluripotency through the reprogramming process [5,6,7], revealing the dynamic nature of induced pluripotency and identifying ten major dynamic expression patterns [8]. Those studies have shown a multistep process that starts with the initial binding of OSKM factors to defined promoters and enhancer regions of the genome, that induces a highly proliferative state and the loss of somatic identity by an early mesenchymal to epithelial transition (MET) [9,10]. This maturation and stabilization phase is characterized by the activation of the core pluripotency circuitry and the silencing of the exogenous transgenes
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