StemCell Therapy

Highlights

Donor age and prolonged cell culture time reduce reprogramming efficiency

Upregulation of the p21 associates with the donor age and time in culture

Knockdown of p21 restored iPSC generation in long-term passaged fibroblasts

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction. Human fibroblasts from 11 different donors of variable age were reprogrammed by ectopic expression of reprogramming factors. Although all fibroblasts gave rise to iPSC colonies, the reprogramming efficiency correlated negatively and declined rapidly with increasing donor age. In addition, the late passage fibroblasts gave less reprogrammed colonies than the early passage cell counterparts, a finding associated with the cellular senescence-induced up regulation of p21. Knockdown of p21 restored iPSC generation even in long-term passaged fibroblasts of an old donor, highlighting the central role of the p53/p21 pathway in cellular senescence induced by both donor age and culture time.

Reprogramming rejuvenates aged somatic cells back into the pluripotent state (Takahashi et al., 2007andTakahashi 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., 2012andYu 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, Trokovic et al., 2014andYu 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., 2010andZhou 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., 2009andSmith 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, Marin et al., 2009andUtikal et al., 2009). Cellular senescence increases with age and one of its hallmarks is the irreversible cell cycle arrest through the activation of the p53/p21 and p16 pathways (Campisi and d'Adda di Fagagna, 2007andNarita 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.

The aim of this report was to evaluate the independent and combined impact of donor age and passage number on the pluripotent reprogramming efficiency of human dermal fibroblasts. Gene expression profiles of selected genes and telomere lengths of starting fibroblasts were analysed in order to identify potential factors behind distinct reprogramming efficiencies. We found that the reprogramming efficiency of human dermal fibroblasts is synergistically affected by donor age and culture time, both inducing cellular senescence through the p53/ p21 pathway.

Donors or their guardians provided their written informed consent for participation. Coordinating Ethics Committee of Helsinki and Uusimaa Hospital District approved generation and use of human iPSC (statement nr. 423/13/03/00/08) on April 2009. Human dermal fibroblasts and foreskin fibroblasts (HFF; CRL-2429; ATCC) were used for reprogramming (Table1).

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Combined negative effect of donor age and time in culture ...

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