To date, all methods to generate induced pluripotent stem cells (iPSCs) require the use of genetic materials and/or potentially mutagenic molecules. Here we report the generation of stable iPSCs from human fibroblasts by directly delivering four reprogramming proteins (Oct4, Sox2, Klf4, and c-Myc) fused with a cell-penetrating peptide (CPP). These protein-induced human iPSCs (p-hiPSCs) exhibited similarity to human embryonic stem cells (hESCs) in morphology, proliferation, and expression of characteristic pluripotency markers. p-hiPSC lines produced with these recombinant proteins were successfully maintained for more than 35 passages and differentiated into derivatives of all three embryonic germ layers both in vitro and in teratomas. This system eliminates the potential risks associated with the use of viruses, DNA transfection, and potentially harmful chemicals and in the future could potentially provide a safe source of patient-specific cells for regenerative medicine.

Over a decade ago, Wilmut and colleagues showed that adult somatic cells could be reprogrammed back to an undifferentiated embryonic state using somatic cell nuclear transfer (SCNT)(Wilmut et al., 1997). However, since that time, attempts to generate patient-specific cells using SCNT have proven unsuccessful (Chung et al., 2009; French et al., 2008). In 2006, a new and less controversial method of reprogramming somatic cells to pluripotency was reported by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006). This and subsequent studies confirmed that mouse and human somatic cells can be reprogrammed to the pluripotent state via viral