LETTERS Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors Jeong Beom Kim 1 *, Holm Zaehres 1 *, Guangming Wu 1 , Luca Gentile 1 , Kinarm Ko 1 , Vittorio Sebastiano 1 , Marcos J. Arau ´zo-Bravo 1 , David Ruau 2 , Dong Wook Han 1 , Martin Zenke 2 & Hans R. Scho ¨ler 1 Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcrip- tion factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1–11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring 2 and that retroviruses them- selves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to develop- ment of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors. Mouse and human somatic cells can be reprogrammed into iPS cells by the expression of a defined set of factors (Oct4, Sox2, c-Myc and Klf4, as well as Nanog and human LIN28) 1–11 . It is possible to generate iPS cells from mouse and human fibroblasts in the absence of c-Myc retrovirus 6,7 , and therefore it was suggested that endogenous expression of c-Myc could have a role in the reprogramming process. Neural stem cells (NSCs) endogenously express Sox2, which may function in maintaining the stemness and multipotency of NSCs 12,13 , and Sox2 was suggested in maintaining cellular pluripo- tency by regulating the expression of Oct4 (ref. 14). NSCs were estab- lished from adult Oct4–GFP (OG2)/ROSA26 heterozygous transgenic mouse brains 15–17 , expressing green fluorescent protein (GFP) under the control of the Oct4 promoter (Oct4–GFP) and the Escherichia coli lacZ transgene from the constitutive ROSA26 locus (ROSA26 lacZ). The established NSCs had the capacity for self-renewal and multipotency (Supplementary Fig. 1). Compared to embryonic stem cells (ESCs), expression of Sox2 was approximately twofold higher in NSCs. c-Myc and Klf4 were also expressed at levels about tenfold higher and eightfold lower in NSCs than in ESCs, respectively (Fig. 1a). Western blot analyses showed that the relationship between protein and RNA levels in NSCs corresponded to that in ESCs for Oct4, Sox2 and Klf4; the c-Myc protein level was comparable in NSCs and ESCs (Fig. 1b). In this study, we attempted to reprogram NSCs into iPS cells by introducing different combinations of the four factors Oct4, Sox2, c-Myc and Klf4 (Supplementary Table 1) using the retroviral MX vector system. We assessed the ability of 15 different transcription factor combinations to induce Oct4–GFP-positive colony formation. Six combinations were able to induce the generation of iPS cells from NSCs, as judged by the formation of GFP 1 colonies and the estab- lishment of iPS cell lines. We observed GFP 1 cells 4 days after trans- duction with the combination containing all four factors—that is, the control combination—and noted a gradual increase in the number of GFP 1 colonies during the first 2 weeks post-infection (Supplementary Fig. 2a). We established four-factor iPS cells from GFP 1 ESC-like colonies on day 14. These four-factor iPS cells stained positive for stage-specific embryonic antigen-1 (SSEA-1) and alkal- ine phosphatase (Supplementary Fig. 2b), showed messenger RNA expression patterns similar to those in ESCs (Fig. 2a, Supplementary Fig. 2c), and led to teratoma formation on injection into nude mice (Supplementary Fig. 2d). Our results demonstrate that NSCs can be reprogrammed into iPS cells by the four transcription factors: Oct4, Sox2, c-Myc and Klf4. Three different combinations of three factors were also capable of generating iPS cells from NSCs: Oct4, Klf4 and c-Myc (OKM); Oct4, Klf4 and Sox2 (OKS); and Oct4, c-Myc and Sox2 (OMS; Supplementary Table 1). We did not observe GFP 1 colonies for the three-factor combinations that did not include Oct4. GFP 1 col- onies were observed 1 week after transduction with the OKM com- bination (without Sox2). However, GFP 1 colony formation was observed only after 14–21 days with the OKS combination (without c-Myc), and was even more delayed with the OMS combination (without Klf4; Supplementary Table 1). Nonetheless, these OKM, OKS and OMS three-factor iPS cells had similar gene expression profiles to ESCs (Supplementary Fig. 3a), and all types of three-factor iPS cells differentiated into all three germ layers (Supplementary Fig. 3b). Taken together, these results indicate that three-factor iPS cells could be generated in the absence of Sox2, Klf4 or c-Myc retroviruses in NSCs, which endogenously express these three factors. We next assessed the ability of two-factor combinations to induce the generation of iPS cells from NSCs. Only two combinations were successful in reprogramming NSCs. We first observed GFP 1 colonies in NSC cultures infected with Oct4 and Klf4 (OK) and 1–2 weeks later in those infected with Oct4 and c-Myc (OM; Supplementary Table 1). The two-factor OM iPS cells showed an ESC-like gene expression pattern and contributed to the three germ layers in teratomas (Supplementary Fig. 3a, b). Klf4 and c-Myc may exert similar func- tions. It is known that Klf4 has a role in the inactivation of the p53 (also known as Trp53) tumour-suppressor gene, which leads to cell immor- talization; Klf4 also works in conjunction with the RAS V12 oncogenic signal transduction protein to stimulate cellular proliferation 18,19 . *These authors contributed equally to this work. 1 Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Ro ¨ntgenstrasse 20, 48149 Mu ¨nster, NRW, Germany. 2 Institute for Biomedical Engineering, Department of Cell Biology, RWTH Aachen University Medical School, Pauwelsstrasse 30, 52074 Aachen, NRW, Germany. doi:10.1038/nature07061 1 ©2008 Macmillan Publishers Limited. All rights reserved