br Materials and methods br Results and discussion

Materials and methods
Results and discussion
Acknowledgments
Introduction Reprogramming rejuvenates aged somatic cells back into the pluripotent state (Takahashi et al., 2007; Takahashi and Yamanaka, 2006). The developmental plasticity of induced pluripotent stem cells (iPSC) demonstrated the potential for regenerative therapies of human diseases (Braam et al., 2013; Song et al., 2012; Yu et al., 2012). Various types of somatic cells have been successfully used for iPSC derivation, including for instance skin fibroblasts, blood cells and myoblasts (Seki et al., 2010; Trokovic et al., 2013, 2014; Yu et al., 2007). Alternative methods for iPSC derivation have been intensively developed to avoid the integration of transgenes, including reprogramming induced by Sendai virus, mRNA, episomal vectors or small molecules (Hou et al., 2013; Nishimura et al., 2011; Warren et al., 2010; Zhou et al., 2009). Although methods for iPSC derivation have been intensively developed, most current technologies are still inefficient, which may be due to intrinsic barriers in the ability of cells to undergo a rapid shift in their proliferative rate (Hanna et al., 2009; Smith et al., 2010). Multiple factors are known to contribute to the efficiency of iPSC generation (Park et al., 2014). For example, differentiation state of the starting cell is a significant factor, since progenitors and stem cells give higher reprogramming efficiency than terminally differentiated cells (Eminli et al., 2009). There is also evidence for varying efficiency for different types of somatic cells from the same donor (Streckfuss-Bomeke et al., 2013). In addition cellular senescence has been shown to affect the reprogramming efficiency (Banito et al., 2009; Kawamura et al., 2009; Li et al., 2009; Marión et al., 2009; Utikal et al., 2009). Cellular senescence increases with age and one of its hallmarks is the irreversible mCAP arrest through the activation of the p53/p21 and p16 pathways (Campisi and d\'Adda di Fagagna, 2007; Narita et al., 2003). These findings suggest that intrinsic properties of somatic cells determine the reprogramming efficiency. Donor age has been shown to have an effect on reprogramming efficiency of murine cells (Wang et al., 2011). Contrary to what has been observed in mice, donor age was suggested not to impair the reprogramming efficiency of human cells (Somers et al., 2010) and iPSC have been successfully derived even from the fibroblasts of centenarians (Lapasset et al., 2011). However, there are no reports on the combined effect of age and culture time on reprogramming efficiency of human cells.
Materials and methods
Results and discussion Pluripotency can be induced from somatic cells by the forced expression of the reprogramming factors OCT4, SOX2, KLF4 and c-MYC. To investigate the effects of donor ages on iPSC reprogramming, we studied human dermal fibroblasts from skin biopsies of 11 individuals representing different ages (0–83years). The donors were either healthy volunteers or patients suffering from neonatal onset diabetes mellitus (PNDM) (Table 1). Although unlikely, we cannot exclude the possibility that the mutations causing PDNM may have had an effect on the reprogramming efficiency. The fibroblast outgrowths (passage 0) were cultured to establish cell lines for further experiments. Fibroblasts at early passage (p6) were transduced with retroviruses encoding OCT4, SOX2 KLF4 and c-MYC, and culture conditions were changed as shown (Fig. 1A). iPSC colonies emerged by day 10 after the induction (Fig. 1B). To compare iPSC-reprogramming efficiency among various donor ages (0–83years), the emerging iPSC colonies with clear hESC morphology and positive for alkaline phosphatase (AP) staining were counted (Fig. 1B–D). Significantly, reprogramming efficiency correlated negatively and declined rapidly with increasing donor age (Fig. 1C and D).