1646 because of strong ethical concerns. MiR-302 is a 23-ribonu- cleotide microRNA (miRNA) expressed abundantly in human ESCs but is absent in all differentiated tissue cells [9]. Despite its presence in ESCs, how does a small RNA, incapa- ble of encoding any protein or peptide, play such a pivotal role in regulating SCR? It turns out that miR-302 functions as a gene silencer and simultaneously downregulates multiple key epigenetic regulators, including lysine-speciﬁc histone demethylases 1 and 2 (namely AOF2/1, LSD1/2, or KDM1/ 1B), DNA (cytosine-5-)-methyltransferase 1 (DNMT1), and methyl-CpG binding proteins 1 and 2 (MECP1/2) [5, 6]. Silencing of these epigenetic regulators induces global DNA demethylation, the ﬁrst sign of SCR. Like a lock, DNA meth- ylation sets up different somatic gene expression patterns in cells and deﬁnes the cells’ properties; the key to SCR, global demethylation, unlocks and resets these differentiated gene expression patterns to a highly uniform ESC-like proﬁle. This ‘‘unlocking’’ of a genome allows transcription machinery access to the ESC-speciﬁc genes and is required for iPSC formation. None of the previously deﬁned transcription factors pro- vided an explanation for the process of global DNA demethy- lation. To clarify this unsolved mechanism, Lin’s studies revealed that miR-302 binds to the gene transcripts (mRNAs) of AOF2/1 and MECP1/2 and forms a RNA-induced silencing complex with argonaute proteins and Dicer endoribonucleases to repress the translation of these important epigenetic regula- tors [5, 6]. In the absence of AOF1 protein synthesis, germ cells do not undergo de novo DNA methylation during oogen- esis [10], while AOF2 deﬁciency causes embryonic lethality due to a progressive loss of genomic DNA methylation and therefore lack of cell differentiation [11]. Hence, silencing of AOF1 and/or 2 is sufﬁcient to induce global demethylation. Silencing of MECP1/2 further enhances this demethylation effect of AOF1/2 deﬁciency [5, 6]. In addition to global demethylation, Lin’s studies also found that miR-302-targeted AOF2 silencing can destabilize DNMT1 activity and prevent replication-dependent DNA methylation during iPSC division, resulting in a passive demethylation mechanism [6]. Based on the analytic results of online miRNA-target prediction program provided by the European Bioinformatics Institute EMBL-EBI (http://www. ebi.ac.uk/enright-srv/microcosm/cgi-bin/targets/v5/detail_view. pl?transcript_id ¼ ENST00000359526), miR-302 also directly targets DNMT1 for gene silencing. Given that DNMT1 func- tions to maintain inherited CpG methylation patterns by meth- ylating the newly replicated DNA during S phase of the cell cycle, deﬁciency of its activity has been shown to cause pas- sive DNA demethylation in early zygotic cells during embry- onic development [12–15]. Taken together, all of the above studies indicate that SCR involves a passive global demethy- lation mechanism. However, this passive demethylation model will generate two hemimethylated cells in every single iPSC colony, an event that has not yet been reported. Passive deme- thylation is unable to remove the methylated sites originally left in the somatic genome before SCR; therefore, only the newly divided daughter cell genomes are demethylated and reprogrammed. Whether these hemimethylated cells are quickly degraded via programmed cell death (apoptosis) dur- ing SCR or further demethylated by an active mechanism remains to be determined. Global demethylation is not a new biological event in view of natural embryonic development. Global demethyla- tion occurs twice during development: ﬁrst during migration of primordial germ cells (PGCs) into the embryonic gonads (approximately embryonic day E10.5 to E13.5) and next in the one-to-eight-cell-stage zygotic cells after fertilization Mechanism of iPSC Generation [13, 14, 16, 17]. Parental methylation imprints are erased and re-established in germline PGCs but largely preserved in post- fertilized zygotic cells [18–20], indicating that the germline and zygotic demethylation mechanisms are not identical. Recent discovery of global demethylation in iPSCs reveals a new kind of artiﬁcial SCR demethylation mechanism compa- rable to the natural ones [6]. Similar to zygotic demethylation, SCR demethylation triggers a massive erasure of genomic methylation sites but preserves parental imprints (Fig. 1). However, unlike zygotes, somatic cells do not receive paren- tal elements from oocytes or sperms, such as paternal prot- amines, maternal proteins, and RNAs. As a result, some zygotic components required for the completion of epigenetic reprogramming during early embryogenesis are missing in SCR. This observation is further supported by recent evidence that the reprogramming induced by SCNT results in more similar epigenomic modiﬁcation and transcriptomic expression patterns to those of ESCs than does the direct reprogramming by four previously deﬁned factors [21]. Prolonging iPSC cul- ture or repeating the differentiation–reprogramming process may improve the clearance of somatic methylation residuals in iPSCs, yet their pluripotency is still not better than that of SCNT-induced stem cells. Therefore, iPSCs may present cer- tain defects due to their lack of some zygotic components. Given that these zygotic components originate in parental germline cells, recent ﬁnding of iPSC-derived PGC-like cells that can be processed through germline demethylation may lead to a solution to this problem [22]. After the revelation of miR-302-mediated global demethy- lation, the next question is how this mechanism relates to the reprogramming induced by previously deﬁned four factors, Oct4, Sox2, Naong, and Lin28. Klf4 and c-Myc are not pre- ferred in the process of iPSC generation due to their potential oncogenic activities. Global demethylation has been reported to promote Oct4–Nanog overexpression in mouse embryos and mouse–human fused heterokaryons [23, 24]. Lin’s studies further demonstrated that elevated miR-302 expression to over 1.1–1.3-fold of the normal human ESC level ( 0.9–1.0 million copies per ESC) triggers both global demethylation and coexpression of Oct4, Sox2, and Nanog in human iPSCs [6, 25]. The expression of Lin28 and many other ESC marker genes was observed 1–3 days later than the presence of Oct4– Sox2–Nanog elevation. A similar miR-302 transfection approach was also shown to increase Oct4 and Nanog expres- sion by twofold in human ESCs [26]. Further research in these ESCs revealed that miR-302 directly silences nuclear receptor subfamily 2, group F, number two, a transcriptional repressor against Oct4 expression, to activate Oct4 transcrip- tion [27]. These ﬁndings have clearly conﬁrmed that global demethylation activates ESC-speciﬁc gene expression in par- ticular, Oct4, Sox2, and Nanog. Through silencing of AOF1/ 2, MECP1/2, and DNMT1, we now understand that miR-302 induces global demethylation and subsequently leads to Oct4– Sox2–Nanog activation essential for the initiation of SCR to form iPSCs. In addition, Oct4 and Sox2 have been found to be the transcriptional activators for miR-302 expression [28–30]. As shown in Figure 2, the mutual stimulation between miR-302 and Oct4–Sox2–Nanog forms a positive feedback regulation loop to maintain the pluripotent status of reprog- rammed iPSCs. This ﬁnding explains why miR-302, Oct4, Sox2, and Nanog are all essential markers for human ESCs. How can we test the interaction between miR-302 and global demethylation? As global DNA demethylation occurs in the cell nucleus while miR-302 functions in the cytoplasm, SCNT is the best method to demonstrate this epigenetic reprogramming mechanism. SCNT is a well-established tech- nology to generate ESC-like pluripotent stem cells by