Induced pluripotent stem cells (iPSCs) can be generated from somatic cells by ectopic expression of defined transcription factors (TFs). However, the optimal cell type and the easy reprogramming approaches that minimize genetic aberrations of parent cells must be considered before generating the iPSCs. This paper reports a method to generate iPSCs from adult human adipose-derived stem cells (hADSCs) without the use of a feeder layer, by ectopic expression of the defined transcription factors OCT4, SOX2, KLF4 and C-MYC using a polycistronic plasmid. The results, based on the expression of pluripotent marker, demonstrated that the iPSCs have the characteristics similar to those of embryonic stem cells (ESCs). The iPSCs differentiated into three embryonic germ layers both in vitro by embryoid body generation and in vivo by teratoma formation after being injected into immunodeficient mice. More importantly, the plasmid DNA does not integrate into the genome of human iPSCs as revealed by Southern blotting experiments. Karyotypic analysis also demonstrated that the reprogramming of hADSCs by the defined factors did not induce chromosomal abnormalities. Therefore, this technology provides a platform for studying the biology of iPSCs without viral vectors, and can hopefully overcome immune rejection and ethical concerns, which are the two important barriers of ESC applications.