Another important advantage of this technical

Another important advantage of this technical report is the successful application of dCas9 activators for validation of PAX7 knock-in reporter Neuronal Signaling Library (Cheng et al., 2013). By design and assembly of site-specific dCas9 activators for PAX7 in human iPS cells, these activators have been screened for induction efficiency. As our data indicated, reporter lines have been successfully validated for reporter activity of PAX7 using this approach. Therefore, we not only verified proper reporter activity of the selected clones, we have also demonstrated the efficiency of dCas9 activators for PAX7 gene induction in human iPS cells. This approach may be also used for directed differentiation of human iPS cells toward early PAX7 positive myogenic progenitors.
Conclusions
Authors\' contributions
Disclosure of potential conflicts of interest
Acknowledgments
Introduction Oral Squamous Cell Carcinoma (OSCC) is one of the most prevalent subtype of Head and Neck Cancers worldwide afflicting >300,000 people annually, with ~150,000 deaths. (http://globocan.iarc.fr/Pages/fact_sheets_population.aspx) (Felthaus et al., 2011). The 5-year survival rate for the disease has not improved through decades, despite advances in treatment modalities that predominantly include surgery and sometimes chemo-radiotherapy (Davis et al., 2010; Chikamatsu et al., 2012). The major cause of failure to cure OSCC includes resistance to therapy, recurrence and metastasis, both local and distant (Mitra et al., 2011). Most of this phenomenon is attributed to the diverse architecture of this tumour that consists of functionally heterogeneous lineages of cancer cells (Kreso and Dick, 2014; Sayed et al., 2011). In this context, role of Cancer Stem Cells, a self-renewing subpopulation of tumour cells that sustain the long-term clonal growth of cancers, is evident (Magee et al., 2012; Clarke et al., 2006). The CSCs escape chemotherapy and are responsible for the recurrence of even more aggressive tumours (Valiyaveedan et al., 2015; Chen, 2009). This hypothesis has gained support in a variety of tumours in addition to cultured cancer cell lines that are shown to harbour CSC-like cells (Chaffer et al., 2013; Yeung et al., 2010; Iacopino et al., 2014). It is advantageous to use malignant cell lines as they are not contaminated with normal stem cells or cancer associated stroma and available in large numbers. Hence cell lines serve as a promising model for exploring the biology of CSCs in multiple cancers. Defining CSC with specific markers has become difficult due to its frequent phenotypic transitions (Clevers, 2011; Shah et al., 2014). Various markers independently or in combination have been investigated to study CSCs from various tumours. These include a series of CSC-entity specifying molecules based on surface marker expression, drug transporters, enzymatic activity, signalling pathways and so on (Routray and Mohanty, 2014; Major et al., 2013). A couple of studies evaluated ALdh1 activity as a functional marker for isolation of CSC-like cells in OSCC (Zou et al., 2012). It was also found to be a relevant CSC marker in closely related Esophageal SCC along with Bmi1 and Nanog (Hwang et al., 2014). The “Side Population” analysis was equally effective in sorting CSCs based on their ability to efflux out Hoechst 33,342 dye (Yanamoto et al., 2011; Golebiewska et al., 2011). Also, CSCs could be simply enriched by growing the cancer cells in serum free media under non-adherent conditions (Chiou et al., 2008). Drug treatment at intermittent and incremental doses has also been shown to successfully expand the CSC population (Xu et al., 2015). In addition, activation of the EMT programme has increasingly been shown to generate CSCs (Ansieau, 2013). Nonetheless, the utility of surface markers, remained the most popular of all isolation and characterization strategies. Prince et al. have proposed CD44+ as the tumour-initiating stem cell in Head and Neck Cancers that could re-establish the original tumour heterogeneity (Prince et al., 2007). Besides, other markers like CD29,CD133, CD24, CD166, and EpCAM in different types of cancers are also used (Keysar and Jimeno, 2010; Zhang et al., 2009; Hurt et al., 2008). CD24, a small membrane glycoprotein is of considerable importance in breast cancer and has recently emerged as a major determinant of stemness in various cancer (Sheridan et al., 2006; Jaggupilli and Elkord, 2012). Although evidences in breast cancer firmly established CD44highCD24low cells to be stem-like, studies in HNSCC have not been conclusive. Here we revisit the prospects of CD44 and CD24 in defining the Cancer Stem Cell markers of OSCC.