Mature adipocytes are terminally differentiated somatic cells. Here, we report the successful generation of induced pluripotent stem （iPS） cells from mouse mature adipocytes by forced expression of six transcription factors （Oct4, Sox2, c-Myc, Klf4, Rarγ, and Lrh1） with a piggyBac transposon-based strategy. The resulting iPS cells were pluripotent as evidenced by the fact that they stained positive for alkaline phosphatase, expressed high levels of key pluripotency markers including Oct4, Nanog, and SSEA1, and remained pluripotent on a 2i media. In vitro differen- tiation of the iPS cells showed that the cell derivatives of all three germ layers could be readily obtained through forma- tion of embryoid bodies. Most importantly, these adipocyte- derived iPS cells were capable of producing chimera with high frequencies when reintroduced into early-stage em- bryos and transmitted through the germ line. This study demonstrates that the new six-factor reprogramming tech- nology facilitates the reset of the terminally differentiated adipocytes to the ground state of pluripotency, enabling us to fully explore the potential of mature adipocytes as a viable cell source for regenerative medicine.
Nanog is a transcription factor identified by its ability to maintain the self-renewal of ES cells in the absence of leukemia inhibitory factor (LIF). Nanog protein contains an N-terminal domain (ND), a DNA-binding homeobox domain (HD) and a C-terminal domain (CD). We previously reported that the CD in Nanog is a transcriptional activation domain essential for the in vivo function of Nanog. Here we demonstrated that the ND in Nanog is also functionally important. Deletion of the ND reduces the transcriptional activity of Nanog on either artificial reporters or native Nanog promoters. This truncated Nanog is also less effective in regulating the endogenous Nanog target genes. Furthermore, the ND truncation disrupted the ability of Nanog to maintain ES cell self-renewal as well. We found that the ND is not required for the nuclear localization of Nanog. These results suggest that the regulation of endogenous pluripotent genes such as oct3/4 and rex-1 is required for the in vivo function of Nanog.
Mouse NANOG plays a critical role in maintaining self-renewal and pluripotency of embryonic stem cells.Yet,the precise mechanism of how mNANOG functions is still less known.Here,we report that mouse NANOG has two nuclear localization signals(NLS,RKQKMR and RMKCKR) which are responsible for the nuclear localization and transcriptional activity in the conserved homeobox domain.NLS mutants of mouse NANOG generate 3 mutants that are localized throughout the cells and lose the transactivation function.We further prove that all three NLS mutants may interact with the wild-type mouse NANOG like NANOG dimerization itself and inhibit the wild-type mouse NANOG activity,acting as dominant negative mutants.The NLS mutants of mouse NANOG may also inhibit activity of oct4 promoter in pluripotent cells,indicating that the NLS mutants can affect the endogenous mouse NANOG function in vivo.These data suggest that the NLS mutants of mouse NANOG may be used as a tool to regulate NANOG activity in pluripotent cells.
In 2006,an article published in Cell by Shinya Yamanaka took by surprise the stem cell research community. By performing systematic retroviral transduction of factors enriched in embryonic stem (ES) cells,the authors demonstrated the reprogramming of mouse fibroblasts into an ES cell-like state. These cells,baptized iPS (induced pluripotent stem) cells,were immediately recognized as a ground-breaking discovery. Subsequently,the same authors and other groups reported a similar achievement with human fibroblasts. Two years later,the number of top quality papers on iPS is astonishing,and interest in the scientific community has risen to a fever pitch. But although iPS has the potential to revolutionize Regenerative Medicine,important questions still remain unanswered. Work from multiple laboratories worldwide including ours is focused on deciphering the molecular mechanisms of iPS,and trying to improve the technique to make it suitable for the clinic. In this review article we briefly discuss the past,present and future of iPS,with emphasis on urgent issues to be solved.
