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    • Here we have extended these

    2018-11-08

    Here we have extended these observations to show that, in addition to maintaining a significantly lower spontaneous mutation frequency than that found in differentiated cells, pluripotent cells are also more resistant to induced mutagenesis than are differentiated cells. Exposure to an EC50 dose of MMS had no effect on mutation frequencies in any of the lines of pluripotent cell types tested, but led to substantial increases in mutation frequencies in all four lines of differentiated somatic cells tested. Our results suggest that pluripotent cells are able to mitigate a moderate mutagenic exposure more effectively than differentiated cell types when cell death rates are similar. This is despite an abbreviated G1 phase of the hif inhibitor in pluripotent cells which actually diminishes the opportunity for post-replication repair of DNA damage in pluripotent cells, thus increasing the need for elevated activity of DNA repair pathways to maintain enhanced genetic integrity in these cells. We previously suggested that differences in maintenance of genetic integrity in pluripotent and differentiated cells reflect differential levels of expression of genetic integrity genes including those involved in the BER pathway (Cooper et al., 2014). Here we directly assessed levels of BER pathway proteins in pluripotent and differentiated cell types, both before and after exposure to mutagen. Our data show that greater resistance to MMS-induced mutagenesis in pluripotent cells correlated directly with higher levels of BER pathway poteins in these same cells. Our results are consistent with a coordinated upregulation of genetic integrity genes in pluripotent cells mediated by mechanisms of epigenetic control (Cooper et al., 2014). This is best exemplified by comparison of mutation frequencies and levels of BER proteins in the TTFs and iPSCs we analyzed, because the latter were derived directly from the former and were, therefore, genetically identical. From this common source, cells maintained in a differentiated state (TTFs) exhibited lower genetic integrity manifest as a higher mutation frequency and lower levels of core BER proteins, whereas pluripotent (iPS) cells exhibited enhanced genetic integrity manifest as lower mutation frequencies and elevated levels of BER proteins. The following are the supplementary data related to this article.
    Acknowledgement This work was supported by the Kleberg Foundation.
    Introduction Allogenic hematopoietic stem cell transplantation (HSCT) is usually complicated with graft-versus-host disease (GvHD) mediated by the donor\'s alloreactive T cells. The immunological responses of these cells against the recipient\'s tissues may lead to damage, primarily in the gastrointestinal tract, liver and skin. Contrary to T cells, NK cells are not involved in GvHD induction and they can even reduce it via the inhibition and lysis of autologous alloreactive GvHD-inducing T cells (Olson et al., 2010; Cerboni et al., 2007). This regulatory role of NK cells may affect T cell activation and proliferation, leading to reduced severity and delayed progression of GvHD following transplant (Alter et al., 2004). Therefore, oppose to GvHD, the graft-versus-leukemia (GvL) effect, mainly supported by the cooperation of cytotoxic T cells and NK cells, benefits transplant recipients by the eradication of residual malignant cells leading to relapse attenuation (Hosseini et al., 2012, 2013, 2015). The potential roles of NK cells in the reduction of post-transplant complications have drawn a significant interest for the use of these cells in cancer immunotherapy. So far, adoptive transfer of NK cells has proven to be an efficient approach for cancer treatment in various animal models (Alici et al., 2007; Basse et al., 2002; Siegler et al., 2005). These findings encourage scientists to develop similar protocols for cancer treatment in the clinical setting using adoptive transfer of either autologous or allogeneic NK cells. However, the isolation of clinical grade NK cells for adoptive immunotherapy is one of the most important obstacles that challenge the safety, feasibility and effectiveness of this therapeutic approach (Sutlu and Alici, 2009). NK cells comprise only a minor fraction of lymphocytes that are identified by their CD3−/CD56+ phenotype. These cells are account for 5% to 15% of circulating lymphocytes and their low number in peripheral blood mononuclear cells (PBMCs) is an obstacle for adoptive transfer of NK cells. To overcome this problem, different methods of isolation and expansion of NK cells have been introduced to obtain a sufficient and effective source of cells for clinical trials (Childs and Berg, 2013). Prior to any clinical setting, some key points about the source of NK cells are the number, purity and state of proliferation and activation of cells. The purity of NK cells can be usually obtained by the depletion of contaminating T cells and NKT cells from PBMCs (Sutlu and Alici, 2009). Typically, simple purification of NK cells with a single-step or two-step procedure is applicable through the isolation of NK cell population from PBMC or other apheresis products that have undergone CD3+ T cell depletion with or without CD56+ selection to increase NK cell purity (Childs and Berg, 2013).