br Results br Discussion Analysis of DNA

Results
Discussion Analysis of DNA-damage foci in CHK1i-treated GSK503 led to the remarkable discovery that CHK1i allowed FA-deficient iPSCs to escape G2 arrest and undergo mitosis without repairing the DSBs that had evidently caused their arrest. A previous study indicates that DSBs arising during G2-M phase are bound together by the MRN complex to be repaired after mitosis (Giunta et al., 2010). The damage is then repaired in G1 by error-prone non-homologous end-joining (NHEJ) or in the subsequent S phase by HR. In the CHK1-inhibited iPSCs, HR is unlikely since CHK1 phosphorylation of RAD51 is required for HR (Sorensen et al., 2005). NHEJ is thus likely to compensate for the FA pathway, but it is predicted to cause chromosomal translocations, which we rarely observed in the FA-deficient iPSCs. Future studies are necessary to determine whether these or other repair pathways compensate for FA in CHK1-inhibited iPSCs. A recent report indicates that BRCA1 is required for ICL-induced activation of CHK1 in FA-deficient cells, indicating that the engagement of the HR machinery upstream of the FA pathway plays a role in checkpoint signaling (Draga et al., 2015). Correspondingly, we observed that HR proteins were engaged at damage foci in G2-phase FA-deficient iPSCs by the localization of RAD51. Since iPSCs are so profoundly dedicated to HR-mediated repair, increased BRCA1 activity may contribute to the hypersensitivity of CHK1 in this cell type. FA is a complex disease that presents with a myriad of congenital abnormalities, indicating that FA affects the development and maintenance of diverse tissues in utero, including but not limited to the hematopoietic system. Fetal or post-natal therapeutic intervention for these developmental defects is not currently available. The conditional FA iPSC model can now provide an experimental platform for discovering new drug targets to sustain stem cell fitness in multiple tissues in the context of FA. CHK1 inhibition is of interest in this regard, given its potential for stimulating the growth of FA hematopoietic cells (Ceccaldi et al., 2011) and current clinical use for the targeting of epithelial tumors, which are common in FA patients. While CHK1 repression bears a risk of introducing mutations through the abrogation of checkpoint control, a therapeutic window should now be explored for the use of CHK1 inhibitors to prevent pre- and post-natal FA phenotypes.
Experimental Procedures
Acknowledgments We would like to thank the Fanconi Anemia Comprehensive Care Center (CCHMC) for access to patient skin biopsies, the Pluripotent Stem Cell Facility (CCHMC) for assistance with iPSC culture and reprogramming, and the Cytogenetic and Molecular Genetics Laboratory (CCHMC) for karyotyping. This work was supported by a Pelotonia Postdoctoral Fellowship and NIH grant R01 CA102357.
Introduction Horses are invaluable animals for companionship and sport. The equine industry creates an estimated economic impact of US$300 billion worldwide, and novel means for addressing equine health issues are constantly required (Tecirlioglu and Trounson, 2007). Musculoskeletal problems, including pathologies or injuries of muscle and cartilage, constitute a leading health threat among horses (Smith et al., 2014). As an example, equine atypical myopathy has been increasingly reported over recent years (Votion and Serteyn, 2008), and equine osteochondrosis is relatively frequent across different breeds (van Weeren and Jeffcott, 2013). Therefore, the quest for novel tools for muscle and cartilage repair is still compelling. In this regard, stem cells may support the needs of veterinary medicine (Cebrian-Serrano et al., 2013). In equine practice, mesenchymal stem cells (MSCs) are commonly used to treat tendinitis and osteoarthritis (Schnabel et al., 2013). However, a comprehensive regenerative approach tailored to both muscle and cartilage is still missing, especially for large-scale applications. Importantly, considering the obvious difficulties of in vivo trials, in vitro models constitute a useful, first-line trial platform for addressing differentiation and heterogeneity of stem cells (Goodell et al., 2015). To this end, induced pluripotent stem cells (iPSCs) hold great potential, in light of their tremendous expansion capacity and wide differentiation potential (Yamanaka, 2009). Recently, iPSCs have been generated from equine fibroblasts (Breton et al., 2013) and keratinocytes (Sharma et al., 2014); however, their differentiation potential toward myocytes or chondrocytes remains unknown. Furthermore, iPSCs tend to retain, at least partially, the intrinsic fate propensity of the cell source (Sanchez-Freire et al., 2014). In mice, iPSCs derived from resident myogenic pericytes, i.e. mesoangioblasts (MABs), show biased myogenic differentiation in both teratoma and in vitro differentiation assays (Quattrocelli et al., 2011). However, it is still unknown whether it is possible to discriminate the intrinsic equine iPSC propensity toward the myogenic and the chondrogenic lineages. To address this question, isogenic settings need to reduce the variability introduced by genetic background (Kotini et al., 2015). Relevantly for putative veterinary applications, the choice of the cell source should be confined to somatic compartments at facilitated reach, e.g. blood and superficial muscle biopsies. Equine peripheral blood has been recently used to isolate circulating progenitors, exhibiting MSC properties (Spaas et al., 2013) and differentiating in chondrocytes (Broeckx et al., 2014). With regard to the muscle, it is still unknown whether equine MABs can be isolated with similar characteristics to murine, canine, or human MABs (Quattrocelli et al., 2014; Sampaolesi et al., 2006), and to which extent equine MABs display myogenic propensity, once cultured in vitro and after reprogramming.