Summary

Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.

Introduction

Embryonic stem (ES) cells, which are derived from the inner cell mass of mammalian blastocysts, have the ability to grow indefinitely while maintaining pluripotency and the ability to differentiate into cells of all three germ layers (Evans and Kaufman, 1981, Martin, 1981). Human ES cells might be used to treat a host of diseases, such as Parkinson’s disease, spinal cord injury, and diabetes (Thomson et al., 1998). However, there are ethical difficulties regarding the use of human embryos, as well as the problem of tissue rejection following transplantation in patients. One way to circumvent these issues is the generation of pluripotent cells directly from the patients’ own cells. Somatic cells can be reprogrammed by transferring their nuclear contents into oocytes (Wilmut et al., 1997) or by fusion with ES cells (Cowan et al., 2005, Tada et al., 2001), indicating that unfertilized eggs and ES cells contain factors that can confer totipotency or pluripotency to somatic cells. We hypothesized that the factors that play important roles in the maintenance of ES cell identity also play pivotal roles in the induction of pluripotency in somatic cells. Several transcription factors, including Oct3/4 (Nichols et al., 1998, Niwa et al., 2000), Sox2 (Avilion et al., 2003), and Nanog (Chambers et al., 2003, Mitsui et al., 2003), function in the maintenance of pluripotency in both early embryos and ES cells. Several genes that are frequently upregulated in tumors, such as Stat3 (Matsuda et al., 1999, Niwa et al., 1998), E-Ras (Takahashi et al., 2003), c-myc (Cartwright et al., 2005), Klf4 (Li et al., 2005), and β-catenin (Kielman et al., 2002, Sato et al., 2004), have been shown to contribute to the long-term maintenance of the ES cell phenotype and the rapid proliferation of ES cells in culture. In addition, we have identified several other genes that are specifically expressed in ES cells (Maruyama et al., 2005, Mitsui et al., 2003). In this study, we examined whether these factors could induce pluripotency in somatic cells. By combining four selected factors, we were able to generate pluripotent cells, which we call induced pluripotent stem (iPS) cells, directly from mouse embryonic or adult fibroblast cultures.