Delivery of Cas protein in an

Delivery of Cas9 protein in an RNP complex with the sgRNA avoids the need for in vivo transcription and translation. Moreover, this approach yields high expression shortly after nucleofection, followed by rapid degradation, thereby facilitating efficient gene editing while minimizing off-target rates. The good performance of the RNP approach in genome-engineering experiments (Liang et al., 2015) may be related to three factors: (1) the possibility to better control the complexing process in vitro, (2) the protection of the Cas9-complexed guide RNA from cellular degradation, and (3) the avoidance of DNA-based cellular toxicity. We found that the combined use of two RNP complexes greatly improves the efficiency of generating targeted translocations in hMSCs, yielding a 12-fold higher rate than the all-in-one plasmid approach. Translocation rates were further improved by co-nucleofecting RNPs and translocation-ssODNs, demonstrating a synergistic effect between these approaches. Surprisingly, translocation rate was not increased by the combined use of RNPs and DNA end-processing enzymes. This might be due to Cas9 RNP degradation preceding expression of the vector-encoded DNA end-processing factors. Although a combination of 3xNLS-RNPs and ssODNs efficiently induced the t(11;22) translocation in hMSCs, this particular translocation was unstable in this cell type, and channel modulator harboring this rearrangement were eventually lost in culture. This finding is in agreement with recent studies of Ewing sarcoma suggesting that MSCs might not represent the target cell-for-transformation in Ewing sarcoma (Kovar et al., 2016; Minas et al., 2016; Renouf et al., 2014; Rodriguez et al., 2011). We therefore attempted to induce the t(11;22) translocation in a less differentiated cell type. We chose hiPSCs because their pluripotent/developmentally early nature as iPSC derivatives constitutes a valid cellular system for disease modeling, including cancer development (Bueno et al., 2011; Menendez et al., 2006; Muñoz-López et al., 2016). Co-nucleofection with RNPs and translocation-ssODNs was able to induce targeted t(11;22) translocations in hiPSCs while maintaining the pluripotent phenotype. These t(11;22)-harboring iPSCs would therefore represent a major step forward in modeling the developmental impact of cancer-associated chromosomal translocations. It allows the investigation of the mechanistic basis and clonal properties of human Ewing sarcoma and would help us to understand the relative contributions of genetic and epigenetic developmental abnormalities to Ewing sarcoma, thus assisting in the integrated design of targeted therapies.
Experimental Procedures
Author Contributions
Acknowledgments This work was supported by funds from the Spanish National Research and Development Plan, Instituto de Salud Carlos III, and FEDER (PI14/01884 to S.R.-P. and PI12/00425 to J.C.C.). R.T.-R. was supported by an international fellowship from Lady Tata Memorial Trust during 2016–2017.
Introduction Glioblastoma (GBM) is the most common and rapidly fatal adult brain tumor. Several developmental pathways important for the growth and proliferation of normal neural progenitors have been shown to be aberrantly reactivated in GBM and glioma stem cells (Canoll and Goldman, 2008; Chen et al., 2012; Lathia et al., 2015; Sanai et al., 2005), through complex genetic and emerging epigenetic alterations (Flavahan et al., 2016; Mack et al., 2015). Among these is the epidermal growth factor receptor (EGFR) pathway, with activating EGFR genomic alterations defining the most common “classical” GBM molecular signature (Brennan et al., 2013; Verhaak et al., 2010) and chromatin remodeling at its promoter driving EGFR overexpression (Erfani et al., 2015). EGFR is also highly expressed in the human developing germinal matrix (GM), as well as focally in the infant and adult subventricular zone (SVZ) (Erfani et al., 2015; Sanai et al., 2011; Weickert et al., 2000), but the stem cell properties and molecular characteristics of human EGFR-positive (EGFR+) neural cells have not been well characterized nor compared with their EGFR+ GBM counterparts, especially in populations derived from fresh human tissues. Here we prospectively isolated EGFR+ cells from fresh GM, SVZ, and GBM human tissues, based on their ability to bind the cognate EGF ligand, which allowed us to directly compare their acute-state functional properties and whole-transcriptome signatures. We demonstrate that developing EGFR+ GM, but not adult EGFR+ SVZ, populations display proliferative stem cell properties in vitro. EGFR+ GBM cells with ligand-binding capacity (LBEGFR+) recapitulate this developmental phenotype functionally in vitro, show capacity for tumor initiation in vivo, and share transcriptomes related to cell growth and cell-cycle regulation.