1648 Mechanism of iPSC Generation Figure 2. Currently established somatic cell reprogramming (SCR) mechanism for induced pluripotent stem cell (iPSC) generation. MiR-302 silences AOF1/2 and DNMT1 activities and, in conjunction with the cosuppression of MECP1/2 and HDAC2, results in global DNA demethyla- tion and chromosomal H3K4me2/3 modiﬁcation. Subsequently, these epigenetic reprogramming events induce ESC-speciﬁc gene expression, in particular Oct4, Sox2, and Nanog, which in turn further stimulate miR-302 expression to form a positive feedback loop cycle crucial for the maintenance of SCR. Based on this mechanistic model, there are two methods for inducing iPSC formation: one is to force the miR-302 expres- sion and the other is to introduce the coexpression of four deﬁned factors (Oct4–Sox2–Nanog–Lin28). Both methods can trigger the activation of this cycling SCR mechanism; however, miR-302 directly induces global demethylation to initiate SCR while the four factors indirectly function through miR-302 expression. Abbreviations: DNMT1, DNA (cytosine-5-)-methyltransferase one; ESC, embryonic stem cell; MECP1/2, methyl- CpG binding proteins 1 and 2; NR2F2, nuclear receptor subfamily 2, group F, number two. epigenetic reprogramming, whereas Oct4, Sox2, and Nanog may indirectly function through miR-302 to continue the reprogramming process. Previous studies have shown that miR-302-induced iPSC generation takes shorter time than does the four-factor induction ( 1–2 weeks vs. 2–3 weeks) [5–7]. Second, delivery of a single small miR-302 is signiﬁ- cantly easier and more efﬁcient than cotransfecting four large transcription factors. The optimal reprogramming efﬁciency reported for miR-302 and four-factor induction methods is >10% and <1%, respectively [3, 4, 6, 7]. Third, some of the previously deﬁned four factors are potential oncogenes, such as c-Myc and Klf4. Based on the analytic results of the online miRNA-target prediction program PICTAR-VERT (http://pictar. mdc-berlin.de/), Klf4 is actually a target of miR-367, a miR-302 homolog naturally expressed in the miR-302 familial cluster. Finally, miR-302-induced SCR is compatible with SCNT technologies. Taken together, miR-302 may replace all previously deﬁned reprogramming factors for safer and more efﬁcient iPSC generation. Five years have elapsed since the initial discovery of iPSC generation to the disclosure of its underlying mecha- nism, which lead to the understanding of the process of ESC- like pluripotent stem cell generation and regeneration. As previous studies were all performed in isolated cells under in vitro conditions, extending these ﬁndings to in vivo applica- tions will be the next challenge. Rejuvenation involves a sys- temic stem cell regeneration process throughout the whole body. This process requires a large stem cell supply and that must be maintained for a relatively long time. Hence, supply REFERENCES 1 Wilmut I, Schnieke AE, McWhir J et al. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385:810–813. and safety of the used stem cells will signiﬁcantly affect the ﬁnal results of rejuvenation. Fortunately, plentiful body cells can now serve as an ideal resource for iPSC generation using miR-302, the introduction of which also prevents tumor for- mation. In view of these current advances in iPSC generation, we found that natural zygotic reprogramming has offered us a solution to the problems of stem cell supply and safety. This solution relies on a miR-302-induced SCR mechanism similar to zygotic reprogramming during early embryogenesis. For modern regenerative medicine, the dual function of miR-302 in both reprogramming and tumor suppression has provided us a convenient means to control the quantity and quality of iPSCs, opening up a new avenue toward the foun- tain of youth. Recent identiﬁcation of the same reprogram- ming and tumor suppression function preserved in the cell lysates of these miR-302-induced iPSCs further leads to a novel SCNT application for iPSC generation without using any transgenic or genomic materials. Based on these ground- breaking discoveries, the development of a feasible strategy and therapy for not only local regenerative medicine but also whole systemic rejuvenation is highly expected in the near future. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conﬂicts of interest. 2 Wakayama T, Perry AC, Zuccotti M et al. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Na- ture 1998;394:369–374. 3 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult ﬁbroblast cultures by deﬁned factors. Cell 2006;126:663–676.