iPS cells can support full-term development of tetraploid blastocyst-complemented embryos.

To our knowledge, for the first time, we demonstrate that induced pluripotent stem cells (iPSCs) can autonomously generate full-term mice via tetraploid blastocysts complementation. Differentiated somatic cells can be reprogrammed into iPSCs by forced expression of four transcription factors—Oct4, Sox2, Klf4, and c-Myc. However, it has been unclear whether reprogrammed iPSCs are fully pluripotent, resembling normal embryonic stem cells (ESCs), as no iPSC lines have shown the ability to autonomously generate full-term mice after injection into tetraploid blastocysts. Here we provide evidence demonstrating that an iPSC line induced by the four transcription factors can be used to generate full-term mice from complemented tetraploid blastocysts and thus appears to be fully pluripotent. This work serves as a proof of principle that iPSCs can in fact generate full-term embryos by tetraploid complementation. Reprogramming of differentiated somatic cells into iPSCs via expression of four defined transcription factors has attracted great scientific and public attention, and the generation of iPSCs from individual patients has raised hopes for treating many degenerative and genetic diseases (Dimos et al., 2008; Ebert et al., 2009; Hanna et al., 2007; Park et al., 2008; Soldner et al., 2009; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2008; Yu et al., 2007). iPSCs can functionally differentiate into all cell lineages within chimeric mice, including germ cells that can ultimately give rise to offspring (Maherali et al., 2007; Okita et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007). However, unlike ESCs or even somatic cell nuclear transfer (SCNT)-produced NT-ESCs, no iPSC lines have so far fulfilled the most stringent pluripotency criterion of being able to generate full-term mice via tetraploid blastocyst complementation (Hayashi and Surani, 2009; Jaenisch, 2008; Maherali and Hochedlinger, 2008; Wernig et al., 2007). To understand the reprogramming mechanisms that occur during iPSC generation, it is important to address whether iPSCs are fully pluripotent. As in previous reports (Brambrink et al., 2008; Stadtfeld et al., 2008), doxycycline (Dox)-controlled Tet-on-inducible lentiviruses carrying cDNAs of the transcription factors Oct4, Sox2, Klf4, and c-Myc were transduced into mouse embryonic fibroblast (MEF) cells from ROSA26-M2rtTA transgenic mice (Figure 1A). After viral transduction, the MEFs were cultured in ES culture medium containing Dox for 12 days until ES-like colonies appeared, and then the Dox was removed from the culture medium (Figure 1B). Four days later, ES-like colonies were individually digested and replated in 24-well plates. After propagation, we selected five iPSC lines that exhibited typical ESC morphology (Figure 1C) for long-term culture and characterization. The ES-like colonies generated were positive for AP activity (see Figure S1 available online). RT-PCR and qPCR analysis demonstrated that the expression of pluripotency marker genes, including Oct4, Sox2, and Nanog, was comparable across all five iPSC lines and normal ESC lines but showed dramatic differences relative to MEFs (Figures 1D and 1E). Silencing of the exogenous lentiviral vectors was also observed (Figure S2). Immunofluorescent staining further confirmed the protein expression of three transcription factors and the surface marker SSEA-1 in the iPSCs (Figure 1F). In addition, other pluripotency-related genes were all expressed at similar levels in the iPSCs and normal ESCs (Figure S3). The karyotypes of all five iPSC lines were normal, with 40 chromosomes (Figure S4). Southern blot analysis was used to examine viral integration into the genome of iPSCs and indicated