TY - JOUR
T1 - Virus-free induction of pluripotency and subsequent excision of reprogramming factors
AU - Kaji, Keisuke
AU - Norrby, Katherine
AU - Paca, Agnieszka
AU - Mileikovsky, Maria
AU - Mohseni, Paria
AU - Woltjen, Knut
PY - 2009/4/9
Y1 - 2009/4/9
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 vectors(1-7). However, such iPS cells contain a large number of viral vector integrations(1,8), any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming(9,10), complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia(11,12). Two transient transfection-reprogramming methods have been published to address this issue(13,14). 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 nonviral 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 transposon(15,16), 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.
AB - 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 vectors(1-7). However, such iPS cells contain a large number of viral vector integrations(1,8), any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming(9,10), complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia(11,12). Two transient transfection-reprogramming methods have been published to address this issue(13,14). 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 nonviral 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 transposon(15,16), 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.
U2 - 10.1038/nature07864
DO - 10.1038/nature07864
M3 - Article
SN - 0028-0836
VL - 458
SP - 771
EP - 775
JO - Nature
JF - Nature
IS - 7239
ER -