Virus-free induction of pluripotency and subsequent excision of reprogramming factors

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Abstract
The discovery that non-germline adult cells can be reprogrammed to become pluripotent, able to differentiate into any cell type, opened up exciting possibilities. Reprogrammed cells — called induced pluripotent stem (iPS) cells — should have great potential in regenerative medicine, but most current methods of producing them involve viral gene delivery that could cause abnormalities in the induced cells. Two groups in this issue report on a collaboration that has succeeded in producing pluripotency in human cells without using viral vectors. Stable iPS cells were produced in both human and mouse fibroblasts using virus-derived 2A peptide sequences to create a multicistronic vector incorporating the reprogramming factors, delivered to the cell by the piggyBac transposon vector. The 2A-linked reprogramming factors, not required in the established iPS cell lines, were then removed. This paper presents a technique to reprogram mouse and human fibroblasts to induce pluripotency. The authors show that the transgene can be removed once reprogramming has been achieved with a piggyBac transposon (described in an accompanying paper; doi:10.1038/nature07863). This system minimizes genome modification in induced pluripotent stem cells and enables complete elimination of exogenous reprogramming factors. Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors1,2,3,4,5,6,7. However, such iPS cells contain a large number of viral vector integrations1,8, any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming9,10, complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia11,12. Two transient transfection-reprogramming methods have been published to address this issue13,14. However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon15,16, we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models.