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

@article{citeulike:4118380, abstract = {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.}, author = {Kaji, Keisuke and Norrby, Katherine and Paca, Agnieszka and Mileikovsky, Maria and Mohseni, Paria and Woltjen, Knut}, citeulike-article-id = {4118380}, citeulike-linkout-0 = {http://dx.doi.org/10.1038/nature07864}, citeulike-linkout-1 = {http://dx.doi.org/10.1038/nature07864}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/19252477}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=19252477}, day = {01}, doi = {10.1038/nature07864}, issn = {0028-0836}, journal = {Nature}, keywords = {reprograming}, month = {March}, number = {7239}, pages = {771--775}, posted-at = {2009-03-06 12:23:53}, priority = {2}, publisher = {Nature Publishing Group}, title = {Virus-free induction of pluripotency and subsequent excision of reprogramming factors}, url = {http://dx.doi.org/10.1038/nature07864}, volume = {458}, year = {2009} }

TY - JOUR ID - citeulike:4118380 L3 - citeulike-article-id:4118380 N2 - 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. IS - 7239 JF - Nature SN - 0028-0836 EP - 775 TI - Virus-free induction of pluripotency and subsequent excision of reprogramming factors PB - Nature Publishing Group VL - 458 SP - 771 KW - reprograming AU - Kaji, Keisuke AU - Norrby, Katherine AU - Paca, Agnieszka AU - Mileikovsky, Maria AU - Mohseni, Paria AU - Woltjen, Knut PY - 2009/03/01/ UR - http://dx.doi.org/10.1038/nature07864 ER -