In addition, a recent study provided additional details of certain epigenetic changes during reprogramming [48••]. As Thy1 (a fibroblast marker) is linearly downregulated and SSEA1 and Oct4 are linearly upregulated during reprogramming, the reprogramming process in this study was roughly divided into three stages: early (day 3, Thy1−), intermediate (days 6–9, SSEA1+), and late (day 12, Oct4+). To determine certain epigenetic profiles in the different stages of reprogramming, PLX3397 solubility dmso ChIP-seq analyses were performed using antibodies against H3K4me3 (an active histone mark) and H3K27me3 (a repressive histone mark)

in cells undergoing reprogramming. It was found that the genes carrying H3K4me3 marks were activated early or gradually (e.g. Fbx15, Cdc25c), whereas genes that were activated late (e.g. Oct4, Nanog) were often either unmarked with H3K4me3 or marked with both H3K4me3 and K3K27me3 in fibroblasts. It was also found that the demethylation of DNA did not happen until the late stage of reprogramming. It was demonstrated that some mouse ESC-specific, cell-cycle-regulating (ESCC) microRNAs, including miR-291-3p, miR-294, and miR-295, could substitute c-Myc and enhance iPSC reprogramming with Oct4/Sox2/Klf4 [49]. Moreover, Subramanyam et al. showed that human

ESCC miRNA orthologs hsa-miR-302b and SB431542 hsa-miR-372 promoted human somatic cell reprogramming through multiple targets, including cell cycle regulators, epigenetic modifiers, and MET regulators [ 50]. In addition to iPSC generation, microRNAs were also shown as powerful regulator for lineage-specific reprogramming. It was reported that miR-9* and

miR-124 were found to directly induce human fibroblasts into neurons with NeuroD2, Ascl1, and Myt1l [ 51]. It was also demonstrated Topoisomerase inhibitor that miR-124 in conjunction with Brn2 and Mytl1 could convert human adult fibroblasts into mature neurons, suggesting that miR-124 plays an important role in neuronal specification [ 52•]. This finding also was supported by recent studies in which knocking down a single RNA-binding, polypyrimidine-tract-binding (PTB) protein could generate mature neurons from mouse fibroblasts via the action of miR-124 [ 53•]. Among these exogenously delivered factors, small molecules and microRNAs, which can be chemically synthesized and do not modify target cell genome, have emerged as powerful tools to manipulate cell fate. While microRNAs offer the advantage of specifically targeting a large number of genes, small molecules provide precise temporal and tunable control over protein function, including rapid and reversible activation and inhibition. With an increased understanding of reprogramming mechanisms and discovery of new molecules, it is conceivable that reprogramming can be achieved in a more efficient and deterministic manner under entirely chemically defined conditions.