Following the successful generation of induced pluripotent stem cells (iPSCs) using transiently expressed reprogramming factors in different combinations (Takahashi & Yamanaka, 2006; Meissner et al., 2007; Wernig et al., 2007; Yu et al., 2007; Nakagawa et al., 2008), a large number of groups reported studies on reprogramming efficiency as well as some molecular mechanisms underlying the reprogramming process (Feng et al., 2009). So far, several chemicals have been shown to enhance the generation of iPSCs. The majority of reported chemicals modulate genome-wide chromatin structure and gene activities (Huangfu et al., 2008a; Mikkelsen et al., 2008; Shi et al., 2008a; Liang et al., 2010). An antioxidant, vitamin C (Vc), and inhibitors of the TGF-β, MEK, and GSK3 pathways also exhibit positive effects on iPSC generation (Shi et al., 2008b; Silva et al., 2008; Ichida et al., 2009; Maherali & Hochedlinger, 2009; Esteban et al., 2010; Li et al., 2010), which suggests that manipulation of the signaling pathways that control cell growth and proliferation may contribute to efficient cell reprogramming.
Rapamycin is a clinically used immunosuppressant that inhibits the mammalian target of rapamycin (mTOR) pathway, involved in cell proliferation, motility, and survival. Rapamycin restores self-renewal in hematopoietic stem cells of aged mice (Chen et al., 2009) and prevents epidermal stem cell exhaustion induced by Wnt-1 expression in mouse skin (Castilho et al., 2009). In the current study, we tested the effects of rapamycin on somatic cell reprogramming. Following the retroviral transduction of four vectors that express Sox2, Klf4, Oct4, and c-Myc (SKOM), primary mouse embryonic fibroblasts (MEFs) from OG2+/−/ROSA26+/− (OG2) mice (containing a transgenic Oct4-GFP reporter) were treated with rapamycin from days 1 to 3 after infection. On day 16 postinfection, we observed the appearance of morphological ES-like and GFP-positive colonies (Fig. 1A, left panel). Rapamycin treatment increased the number of GFP-positive colonies in a dose-dependent manner up to 0.3 nm (Fig. 1A, right panel). FACS analysis of SKOM-infected OG2 MEFs on day 12 postinfection displayed an over 5-fold increase in GFP-positive cells in samples treated with 0.3 nm rapamycin (Fig. 1B). Furthermore, we found that treatment of MEFs with rapamycin on days 1–3 postinfection was more effective in promoting reprogramming than treatment at later times after infection (Fig. 1C). These results suggest that rapamycin acts early in the reprogramming process.
To confirm the pluripotency of iPSCs generated after rapamycin treatment, we randomly picked colonies to establish multiple iPSC lines. All of these lines were morphologically indistinguishable from mouse ES cells, and they stained positive for alkaline phosphatase (Fig. 1D). The endogenous expression of Oct4, Sox2 and Nanog as well as the silencing of exogenous retroviral factors expression was verified by real-time RT–PCR (Fig. 1E,F). A randomly chosen clone (iPS-Rapa#4) was then subjected to further analysis. These cells expressed high levels of the pluripotency markers SSEA1 and Nanog (Fig. 1G), and were karyotypically normal (Fig. 1H). Most importantly, these cells were competent to generate germline chimeras (Fig. 1I). These results suggest that rapamycin treatment does not compromise iPSC pluripotency.
To test whether the reprogramming-enhancing effect of rapamycin was because of its inhibition of the mTOR pathway, we used PP242, another potent mTOR pathway inhibitor (Apsel et al., 2008). PP242 functions as a selective inhibitor that targets the ATP-binding domain of mTOR. Treatment with 0.1 nm PP242 led to a 5-fold increase in the reprogramming efficiency similar to rapamycin (Fig. 2A). These results indicate that inhibition of mTOR activity promotes somatic cell reprogramming.
Rapamycin extends the lifespan in various model organisms (Fontana et al., 2010). Therefore, we tested whether inhibition of the mTOR pathway by PP242 had similar lifespan-extending effects to rapamycin in fruit flies (Fig. 2B,C). Treatment of PP242 significantly extended the lifespan of fruit flies similar to rapamycin (Fig. 2B,C). These results suggest that mTOR might be a common pathway that mediates both longevity and somatic cell reprogramming efficiency.
Similar to the mTOR pathway, the IIS pathway is ubiquitously expressed, and its inhibition is associated with increased longevity and the delayed onset of age-related disorders in diverse species (Fontana et al., 2010). Therefore, we tested whether inhibition of the IIS pathway by PQ401, an inhibitor of IGF1 receptor, could also extend the lifespan and promote somatic cell reprogramming. Indeed, PQ401 treatment led to an almost 4-fold increase in the reprogramming efficiency of the SKOM-infected MEFs (Fig. 2D) and extended the lifespan of flies (Fig. 2E,F). These results further support a correlation between mechanisms regulating longevity and mechanisms regulating reprogramming efficiency.
We then tested the effects of six other longevity-promoting compounds on somatic cell reprogramming, including two sirtuin activators (resveratrol and fisetin), an autophagy inducer (spermidine), a PI3K inhibitor (LY294002), an antioxidant (curcumin), and an adenosine monophosphate-activated protein kinase (AMPK) inhibitor (metformin). We treated SKOM-infected OG2 MEFs with these compounds from day 1 to day 3 postinfection and subjected them to FACS analysis on day 12. Resveratrol, fisetin, spermidine, LY294002, and curcumin enhanced reprogramming to different extents (Fig. 2G and Fig. S1), but the AMPK activator, metformin, did not improve reprogramming efficiency.
Taken together, our results show a functional correlation between the regulation of cell reprogramming and that of organismal longevity. These compounds modulate organismal longevity by targeting different signaling pathways (Fig. 2H). Therefore, further investigation to unravel the molecular mechanisms of their reprogramming-promoting effects would be of importance to understand the functional correlation between the regulation of cell reprogramming and that of organismal longevity. Furthermore, many chemicals that enhance mouse somatic cell reprogramming also have similar positive effects on human somatic cell reprogramming (Huangfu et al., 2008b; Feng et al., 2009; Esteban et al., 2010; Mali et al., 2010; Zhu et al., 2010). Therefore, our findings in mouse cells may also be applicable to human cells, which is under current investigation. It is reported that the reprogramming process can be divided into three phases, namely initiation, maturation, and stabilization (Samavarchi-Tehrani et al., 2010). Studies have shown that TGF-beta inhibitors function at the initiation stage of reprogramming (Li et al., 2010; Samavarchi-Tehrani et al., 2010), whereas MEK inhibitors function at the maturation and stabilization phase (Shi et al., 2008b). Our results show that rapamycin and other longevity-related chemicals function at the initial stage of reprogramming. These chemicals may facilitate the bypass of certain reprogramming barriers, e.g., by preventing cell senescence that is induced by the reprogramming factors (Banito et al., 2009; Li et al., 2009) or by facilitating the mesenchymal-to-epithelial transition (Li et al., 2010; Samavarchi-Tehrani et al., 2010), or by other mechanisms.