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  • Induced pluripotent stem cells have emerged as powerful tool

    2018-11-14

    Induced pluripotent stem nicotinic receptor agonist have emerged as powerful tool for many applications including disease modelling, drug screening and cell based therapies (Takahashi and Yamanaka, 2016). Therefore, increased efficiency of cell reprogramming is beneficial as it could help reduce the time required to produce iPSC. In addition, an enhanced proliferation rate would also be useful as in many applications the number of cells is critical for the successful use of iPSC. Several aspects have been regarded as contributing factors towards reprogramming efficiency including the type and the differentiation stage of the starting adult somatic cells as well as their proliferation rate. Some particular cell types exhibit higher reprogramming efficiency, for example mesenchymal stem cells (MSC) are easier to reprogramme than keratinocytes from the same individual, possibly due to the higher methylation state within key pluripotency genes in the keratinocytes (Streckfuss-Bomeke et al., 2013). Likewise, the differentiation stage of the starting cells is also important: Eminli and colleagues have shown that undifferentiated haematopoietic cells are far more efficiently reprogrammed compared to terminally differentiated B and T cells (Eminli et al., 2009). In addition to these factors, recent observations have also shown that proliferation rate and/or cellular senescence of the starting cells as well as the age of donor cells appear to play essential roles in determining reprogramming efficiency (Utikal et al., 2009; Banito et al., 2009; Kawamura et al., 2009; Li et al., 2009; Marion et al., 2009). We therefore reasoned that modification of the proliferation rate of the starting cells by modulating a key signalling pathway that regulates cell proliferation might be used as a strategy to improve cell reprogramming. We focused on the Hippo signalling since this pathway is one of the primary regulators of cell proliferation, cell survival and organ size control (Yu and Guan, 2013; Johnson and Halder, 2014). Components of the Hippo pathway modulate proliferation in various cell types. For example, Mst1 gene silencing induces proliferation of glioma cells (Chao et al., 2015), whereas in mouse intestinal epithelium ablation of both Mst1 and Mst2 resulted in cell expansion and proliferation (Zhou et al., 2011). The other central components of the Hippo pathway, Lats1/2 are also essential in mediating cell proliferation. Knockout of Lats1 and Lats2 increases proliferation of biliary epithelium and hepatoblasts (Yi et al., 2016), in contrast, overexpression of Lats1 reduces proliferation of human breast cancer cells MCF-7 (Xia et al., 2002). TAZ, one of the downstream effectors of the Hippo pathway, also promotes cell proliferation (Lei et al., 2008). In this study we focused on targeting the Mst1 kinase. In the Hippo signalling cascade Mst1 acts as a negative regulator of the proliferative signal by phosphorylating Lats1/2 and eventually results in YAP phosphorylation and hence inactivation (Yu and Guan, 2013; Johnson and Halder, 2014). In the present study we found that genetic ablation of Mst1 significantly enhanced the reprogramming efficiency of adult mouse skin fibroblasts to iPSC. The improvement of reprogramming efficiency might be, at least in part, due to the increased proliferation rates of the starting somatic cells as the skin fibroblasts lacking Mst1 displayed higher proliferation rates in both normal and starving medium. This is consistent with a previous report showing that highly proliferative early passage mouse embryonic fibroblasts (MEFs) displayed higher efficiency of reprogramming compared to late passage MEFs, which display a lower proliferation rate (Utikal et al., 2009). In agreement with this, various observations have demonstrated that in less proliferative cells or in senescent cells the reprogramming efficiency is low (Banito et al., 2009; Kawamura et al., 2009; Li et al., 2009; Marion et al., 2009). Activation of the p53 pathway in such cells is thought to be the underlying mechanism for the low reprogramming efficiency (Kawamura et al., 2009).