In summary we have generated a stable e

In summary, we have generated a stable, expandable human NSC line (LM-NSC008) by overexpression of the L-MYC gene. We have characterized the LM-NSC008 cell line in vitro and in vivo. Our findings demonstrate the potential therapeutic utility of LM-NSC008 NSC 74859 for therapeutic drug delivery to CNS tumors, restorative therapies for TBI, and possibly other diseases of the CNS. Further pre-clinical studies in CNS disease models are warranted for translational development and eventual clinical use of L-MYC-transduced NSC lines.
Experimental Procedures
Author Contributions
Acknowledgments The authors acknowledge the technical expertise and advice of Sofia Loera of the City of Hope Pathology Core, and the editorial assistance of Dr. Keely L. Walker and Andrea Lynch (City of Hope). We also thank Dr. Shinya Yamanaka for providing the L-MYC plasmids. This work was supported by funding from NIH (R01 NS065069-07), Accelerate Brain Cancer Cure (ABC2), Alex’s Lemonade Stand Foundation, Altschul foundation, and Pediatric Cancer Research Foundation. Research was also supported by work performed in the Bioinformatics, Cytogenetics and Pathology Cores supported by the National Cancer Institute of the NIH under grant number P30CA033572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. K.S.A. and A.J.A. are uncompensated Board Members, Chief Scientific and Chief Financial Officers, and shareholders of TheraBiologics.
Introduction The use of human pluripotent stem cells (hPSCs) in studying human biology and disease requires precise genome-targeting methodologies. While different from mouse cells, gene targeting in either human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) is found to be technically difficult (Capecchi, 2005; Hockemeyer and Jaenisch, 2010; Zwaka and Thomson, 2003). This situation is now much improved with the development of the custom-engineered nucleases (CENs) (Hendriks et al., 2016). CENs are designed to introduce site-specific double-strand breaks (DSBs) within the genome, which trigger DNA repair and therefore facilitate genetic engineering (Hendriks et al., 2016; Hsu et al., 2014). To date three CENs have been developed, among which the type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is increasingly preferred, based on its high efficiency and less laboriousness compared with zinc-finger nucleases and transcription activator-like effector nucleases (Ding et al., 2013b; Hou et al., 2013). The type II CRISPR/Cas9 system relies on three components as follows: the DNA nuclease Cas9, the CRISPR RNA (crRNA), and trans-activating crRNA (tracrRNA) (Jinek et al., 2012). To facilitate laboratory use, a single guide RNA (gRNA), which is encoded by a single vector plasmid (Cho et al., 2013; Cong et al., 2013; Mali et al., 2013), has now replaced the crRNA/tracrRNA duplex. The gRNA contains a 15- to 23-bp target DNA-matching sequence immediately upstream of a protospacer adjacent motif (PAM) (Fu et al., 2014; Hsu et al., 2013; Stemmer et al., 2015). The CRISPR/Cas9 derived from Streptococcus pyogenes is the most widely used system and has a 5′-NGG-3′ PAM (Cong et al., 2013; Mali et al., 2013). The 15- to 23-bp-long complementary sequence binds to the target genomic locus through strict Watson-Crick pairing (Fu et al., 2014; Hsu et al., 2013; Stemmer et al., 2015), where the gRNA directs Cas9 for DNA cleavage and therefore results in a DSB at a desired site (Jinek et al., 2012). A DSB introduced into the genome will initiate DNA repair through either error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) in the presence of exogenous DNA templates (Geisinger et al., 2016; Heyer et al., 2010; Jasin and Rothstein, 2013; Lackner et al., 2015).