Lin 1647 Figure 1. Comparison of natural germline-zygote and manually forced SCR demethylation pathways. Global demethylation occurs naturally in two developmental stages, one during formation and migration of PGCs into the embryonic gonads (germline demethylation) and a second time in one-to-eight-cell-stage zygotes before the morula stage (zygotic demethylation). Parental imprints are erased and re-established during germline demethylation but not in zygotic demethylation, resulting in different genomic imprinting patterns from the parents. On the other hand, SCR demethylation can be forced in somatic cells by manually introducing the expression of either miR-302 or four deﬁned reprogramming factors (Oct4–Sox2–Klf4-c–Myc or Oct4–Sox2–Nanog–Lin28). Notably, Oct4 and Sox2 can also induce miR-302 expression. Because of the effects of miR-302-targeted epigenetic gene silencing, the somatic cell genome is forcedly demethylated without the erasure of parental imprints. As a result, expression of parental germline elements is still inactivated by the imprints. Given that parental elements are essential for normal zygotic development, their deﬁciency may cause developmental defects in SCR-induced iPSCs. Abbreviations: ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells; PGCs, primordial germ cell; SCR, somatic cell reprogramming. transferring a somatic cell nucleus into the MII-stage oocyte cytoplasm [1, 2]. Following the same procedure but instead of oocyte cytoplasm, we transferred somatic cell nuclei into the cytoplasm of denucleated miR-302-induced iPSCs. Most (93%) of the hybrid cells were reprogrammed to iPSC-like pluripotent cells possessing ESC-like properties [6]. Global demethylation was also quickly detected a few days after SCNT, indicating that the reprogramming factors in iPSCs are functionally similar to those found in oocyte cytoplasm. Con- versely, transferring iPSC nuclei into the somatic cell cyto- plasm failed to form any viable cell. Hence, the earliest reprogramming effector, the ‘‘initiator,’’ lies in the cytoplasm rather than nucleus of an iPSC. This ﬁnding is coincident with the previous SCNT results using the oocyte cytoplasm, which contains several miR-302 homologs that may have the same reprogramming function, such as miR-200c and miR- 371–373. Given that miR-302 is a cytoplasmic effector whereas Oct4, Sox2, and Nanog are all nuclear transcription factors, it is conceivable that miR-302 is responsible for ini- tiating SCR through global demethylation in the SCNT- induced iPSCs. On the other hand, the transfer of iPSC nuclei fails to induce pluripotency, suggesting that somatic cyto- plasm may lack a key reprogramming effector required for iPSC generation. Alternatively, somatic cytoplasm may con- tain an inhibitor directed against iPSC formation; however, this possibility has been ruled out by further induction of miR-302 elevation in the hybrid cells containing the miR- 302-induced iPSC nuclei, showing that the efﬁciency of iPSC generation is proportional to the extent of induced miR-302 elevation [6]. Because of this new SCNT approach, miR-302- induced iPSCs may be applied to replace oocytes to avoid potential ethical concerns. Regenerative medicine presents the key to future therapies for treating genetic disorders, physical abnormalities, injures, www.StemCells.com cancers/tumors, and aging. Supply and safety of the stem cells are two of the most important bottlenecks in regenerative medicine. Prior efforts have succeeded in isolating various pluripotent/multipotent stem cells from embryos, umbilical cords/amniotic ﬂuids, and reproductive organs; yet, none of these stem cells can be made in a large quantity enough for patients. Without knowledge of the mechanism underlying stem cell generation, it is difﬁcult to produce sufﬁcient pluri- potent stem cells for regenerative medicine. Following the recent identiﬁcation of miR-302-mediated SCR mechanism (Fig. 2), an induced reprogramming process mimicking the natural zygotic development event, we have now insight into the regula- tory mechanism necessary for stem cell generation and a method of reprogramming somatic cells to ESC-like iPSCs. This SCR mechanism is not only capable of generating sufﬁcient iPSCs but also preventing tumor formation in these iPSCs [25]. During SCR, miR-302 functions to silence both cyclin E-CDK2 and cyclin D-CDK4/6 cell cycle pathways during G1–S-phase transi- tion, consequently preventing iPSC tumorigenicity. Furthermore, miR-302 also silences polycomb ring ﬁnger oncogene BMI1, a cancer stem cell marker, to promote the expression of two tumor suppressor genes, p16Ink4a and p14/p19Arf. The combination of these two tumor suppression effects results in a tightly regu- lated cell cycle rate similar to that of the one-to-eight-cell-stage embryonic cells in early mammalian zygotes (20–24 hours per cycle). As human ESCs express abundant miR-302, this tumor suppressor function of miR-302 may explain how early embry- onic development circumvents tumor formation. As a result, we may use this new ﬁnding for developing tumor-free iPSCs to improve the safety of regenerative medicine. The mechanism of miR-302-induced SCR presents several advantages over the currently established four-factor induction methods. First, miR-302 directly silences its targeted epige- netic genes and induces global DNA demethylation to initiate