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  • There are two major repair

    2018-11-08

    There are two major repair methods for DSB damage repair, namely HRR and nonhomologous end joining (NHEJ). HRR results in error-free repair, but NHEJ sometimes leads to errors. During HRR, DSB damage repair uses a template containing hundreds of order UNC0638 pairs of sequence homology, and usually the sister chromatids are available to serve as templates (Haber, 2000; Morrison et al., 2000; Takata et al., 1998). The S phase of ESCs has a long duration, so most of the ESC genomes would have sister chromatids, which assures the process of HHR (Tichy and Stambrook, 2008). Rad51b has been reported to play an important role in the HRR process to repair DSB, and Rad51b is also a protein kinase that can regulate cell cycle-related genes (Havre et al., 2000; Shrivastav et al., 2008; Stordal and Davey, 2009). In our study, we found that Rad51b is also important for mESCs. Knockdown of Rad51b results in inefficient DNA damage repair and a faster cell cycle, which creates a vicious circle of producing more DSB and SSB damage. We further found that Rad51B can be regulated by the miR-590-Acvr2a pathway. Rad51b can be upregulated by miR-590 or knockdown of Acvr2a in mESCs. Moreover, knockdown of Rad51b attenuated the effects caused by knockdown of Acvr2a or overexpression of miR-590. Our results suggested that miR-590/Acvr2a/Rad51b signaling axis balances the coexistence of DNA damage repair and rapid proliferation, assuring the stabilization of mESCs. Our study provides insights into the regulation of proliferation and DNA damage repair during mESC self-renewal, and it suggests that an ESC-related signaling pathway not only maintains the stabilization of mESC self-renewal or pluripotency but also may be the regulator or responder of DNA damage repair.
    Experimental Procedures
    Acknowledgments
    Introduction Cellular reprogramming has opened new avenues to investigate human disease and identify potential targets for drug discovery (Bellin et al., 2012). This technology is particularly useful for cell types in which the target tissue is not accessible, like the brain. It is now possible to differentiate human embryonic stem (hES) and human-induced pluripotent stem (hiPS) cells into different types of neurons (Hu et al., 2010; Qiang et al., 2014; Velasco et al., 2014; Zhang et al., 2013). However, the generation of neuronal cells from pluripotent stem cells involves long and complex protocols with problematic variability. Alternatively, direct lineage conversion (or transdifferentiation) of somatic cells into neurons (induced neurons [iNs]) has been achieved by forced expression of lineage-specific transcription factors and microRNAs (miRNA) (Ambasudhan et al., 2011; Caiazzo et al., 2011; Pang et al., 2011; Pfisterer et al., 2011; Vierbuchen et al., 2010; Yoo et al., 2011). Using this approach, several cell types (Giorgetti et al., 2012; Karow et al., 2012; Marro et al., 2011) have been converted into functional neurons in vitro and also in vivo (Guo et al., 2014; Su et al., 2014; Torper et al., 2013). However, for delivery of exogenous reprogramming factors, most available protocols have used integrative viral vectors, and the conversion process was rather inefficient. Only recently, nonintegrative methods based on Sendai virus (SeV) or chemically defined culture conditions have been described for the direct conversion of nonhuman cells into neural progenitor cells (iNPCs) (Cheng et al., 2014; Lu et al., 2013). Here, we investigated whether a similar nonintegrative strategy is applicable for the conversion of human hematopoietic cells directly into neurons. Importantly, peripheral blood (PB), which is routinely used in medical diagnoses, represents a noninvasive and easily accessible source of cells for reprogramming both healthy donor and disease-specific patient cells. Based on our previous study (Giorgetti et al., 2012), we chose SOX2 and c-MYC SeV vectors to reprogram CD133-positive cord blood (CB) cells and adult PB mononuclear cells (PB-MNCs). We found that the overexpression of SOX2 and c-MYC by SeV accelerated and increased the efficiency of neural conversion of CD133-positive CB cells (CB-iNCs) when compared with retroviral vectors. SOX2 and c-MYC were also sufficient to convert PB-MNCs into neuronal-like cells (PB-iNCs). However, compared with CB-iNCs, the process was less efficient, and the resulting PB-iNCs showed limited expansion, differentiation capacity, and functional properties. Our results demonstrate the feasibility for rapid and efficient generation of iNCs from CD133-positive CB cells using nonintegrative SeV while underscoring the impact of target cell developmental stage on the reprogramming process for lineage conversion.