Different developmental potential of pluripotent stem cells generated by different reprogramming strategies.

Dear Editor, Recent studies show that induced pluripo-tent stem cells (iPSCs) generated through ectopic expression of transcription factors retain an epigenetic memory of their original somatic cells (Kim et al., 2010; Polo et al., 2010) or aberrant silencing of a single imprinted gene cluster (Liu et al., 2010; Stadtfeld et al., 2010), which affects their developmental and differentiation potentials. In contrast, nuclear transfer can more faithfully reprogramme somatic cells into embryonic stem (ES) cells (nuclear transfer ES cells, ntESCs) 2006). However, it is still controversial whether reprogramming method per se determines the pluripotency of resulting cells. Here, using genetically identical donor cells, we generated three kinds of mouse reprogrammed cells: iPSCs, ntESCs, and iPSC-nt-ESCs, after successively reprogramming of iPSCs by nuclear transfer. We found that ntESCs had better developmental potential compared with iPSCs, and following nuclear transfer can not rescue, but deteriorate the developmental deficiency of iPSCs, resulting in the worst developmental ability in iPSC-nt-ESCs. In order to minimize genetic variations among different reprogrammed cells, we established a genetically homogenous secondary reprogramming system, in which mouse embryonic fibroblasts (MEFs) carrying doxycycline (Dox)-inducible lentiviruses expressing Oct4, Sox2, Klf4, and c-Myc (OSKM-MEFs) were isolated (Huang et al., 2009) and used as donors for different reprogramming experiments. To generate secondary iPS cells, OSKM-MEFs were cultured in mouse embryonic stem cell medium supplemented with Dox, which induced the expression of transgenes and initiated the reprogramming process. After exposing OSKM-MEFs to Dox for 18 days, iPS cell colonies were expanded in the absence of Dox and four iPS cell lines were established from MEF-1 and MEF-2 (iPSC-1 and iPSC-2 generated from MEF-1; iPSC-3 and iPSC-4 from MEF-2; Figure 1A; Supplementary Figure S1A). All iPS cell lines showed an ES-like morphology , exhibited alkaline phosphatase (AP) activity and expressed the pluripotent markers Oct4, Sox2, and SSEA-1 (see Supplementary Figure S1B). Bisulphite sequencing analysis of endogenous Oct4 promoter of iPS cell nuclei revealed that epigenetic state of the somatic cells had been reprogrammed (Supplementary Figure S1C). These results indicated that all iPSCs had activated their endo-genous pluripotency core transcriptional network. To generate genetic identical ntESCs, we performed nuclear transfer (Yang et al., 2010) using the same MEFs as nucleus donors (Figure 1A). From 526 oocytes successfully reconstructed with the nuclei of MEF-1 and MEF-2, we cloned blastocysts and established a set of 12 ntES cell lines (Supplementary Table S1). Next, to test whether successively reprogramming by nuclear …