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

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 vectors1-7. However, such iPS cells contain a large number of viral vector integrations1,8, any one of which could cause unpredictable genetic dysfunction. While c-Myc is dispensable for reprogramming9,10, complete elimination of the other exogenous factors is also desired since ectopic expression of either Oct4 or Klf4 can induce dysplasia11,12. Two transient transfection reprogramming methods have been published to address this issue13,14. However, the efficiency of either approach is extremely low, and neither has thus far been applied successfully to human cells. 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 chimeric 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.

Mechanism and methods to induce pluripotency

“…To meet this need, several nonintegration systems and removable systems have been developed. These include episomal vectors (Yu et al, 2009), the piggyBac transposon system (Kaji et al, 2009;Woltjen et al, 2009;Yusa et al, 2009), and minicircle vectors (Jia et al, 2010).…”

“…Although iPSCs can be obtained by removable transposon or the non-integration episomal system, all of these methods still involve introducing foreign DNA into the cell so that the resulting cell lines need to be sequenced to verify that they are indeed free of genome alteration (Kaji et al, 2009;Woltjen et al, 2009;Yu et al, 2009). Thus, a reprogramming approach that does not use any DNA is highly desirable.…”

Klf4 Interacts Directly with Oct4 and Sox2 to Promote Reprogramming

“…Additionally, inducible systems have been developed to better control transgene expression [7]. Novel methods requiring no viral integration have also been developed [8][9][10][11][12], as have strategies using small molecules to promote reprogramming efficiency [13][14][15].…”

Differentiation of Stem Cells into Insulin-Producing Cells: Current Status and Challenges

“…In addition, the viral vectors after integration with the host may lead to unexpected genetic disorder (Okita et al 2007;Takahashi et al 2007;Tateishi et al 2008;Yu et al 2009). Therefore, new and improved methods of generating iPS cells, including minimal or no genetic modification are needed (Huangfu et al 2008;Kaji et al 2009;Okita et al 2008;Stadtfeld et al 2008;Woltjen et al 2009;Yu et al 2009;Zhou et al 2009). Tateishi et al (2008) reported that iPS cells derived from human skin fibroblast by retroviral expression of OCT4, SOX2, c-MYC, and KLF4 have the potential to differentiate into insulin-producing islet-like clusters (ILCs).…”