The pig has many advantages over

The pig has many advantages over mouse models for pre-clinical studies involving hESC-CPC transplantation, namely its similar heart size and physiology to human. In addition, pig cardiomyocytes have been shown to exhibit similar contraction rates and analogous action potential duration to humans (Stankovicova et al., 2000). We sought to determine whether CD13+/ROR2+ pre-cardiac mesodermal mps1 could engraft into the porcine myocardium and further differentiate into cardiovascular lineages. We chose to deliver 13R2+ cells into uninjured pig hearts to eliminate the many variables associated with the injury process. Extensive ICC analysis was performed, as in this context the NKX2-5eGFP and αMHC-mCherry fluorescence could not be reliably distinguished from the highly autofluorescent background. Our results indicated that many transplanted 13R2+ cells survived, engrafted, and differentiated toward definitive cardiovascular cell types in the pig heart after approximately 6 weeks. We observed small vessels that incorporated 13R2+-derived endothelial and vascular smooth muscle cells. In addition, we identified numerous areas within the pig\'s heart containing 13R2+-derived cardiomyocyte clusters ranging from 5 to >1,000 cells. Interestingly, many of these cells had organized sarcomere and formed Connexin-43 junctions between adjacent grafted cells. Whether 13R2+ cells are able to structurally and functionally integrate into the host myocardium and offer a therapeutic benefit to injured pig heart warrants further investigation. Such studies will be particularly important, given there is a negligible difference between the engraftment potential of hESC-derived cardiomyocytes and cardiac mesoderm (Chong et al., 2014). Ultimately, it is possible that combinations of different cell types (i.e. cardiomyocytes and cardiac progenitors) may improve graft survival and functional outcomes (Xiong et al., 2012; Ye et al., 2014).
Experimental Procedures
Acknowledgments The authors would like to thank all the members of the Ardehali laboratory for instructive discussions and suggestions. The authors would also like to thank the UCLA TRIC center and DLAM, namely Anthony Smithson, Sandra Duarte Vogel, and Janlee Jensen, for all their support and expertise in the handling of large animal models. Thanks are also due to the UCLA Flow Core, Namely Jessica Scholes and Felicia Codrea. Furthermore, the authors would like to especially thank Armin Hojjat, and Drs. Gay Crooks, Hanna Mikkola, and Thomas Vondriska for their valuable input and guidance. This work was supported in part by grants from the California Institute of Regenerative Medicine (CIRM) (RC1-00354-1) (R.A.) and Eli & Edith Broad Center of Regenerative Medicine and Stem Cell Research Center at UCLA Research Award (R.A.).
Introduction CRISPR-Cas9, an emerging genome surgery tool, exploits an engineered ribonucleoprotein complex consisting of two essential components: (1) a protein, Cas9; and (2) a single-guide RNA (sgRNA). Together, the Cas9-sgRNA complex cuts a specific target sequence in the genome. Human cells and tissues edited by CRISPR-Cas9 are important resources for drug target identification (Kasap et al., 2014; Shi et al., 2015; Smurnyy et al., 2014), regulatory science (Hsu et al., 2014), medicine (Doudna, 2015), and basic biology (Hsu et al., 2014; Sternberg and Doudna, 2015). However, human gene-editing experiments frequently require laborious cloning of expression plasmids for each sgRNA, and there are limited opportunities in these culture systems to watch and perturb genome surgery in action, as it is difficult to isolate and image living mutant cells during and shortly after the DNA cleavage event. Overall, there is a need to expand the throughput and capabilities of current in vitro human culture systems where novel genome surgery approaches can be evaluated with human cells and tissues (Baltimore et al., 2015). Advanced capabilities with human pluripotent stem cells in particular could eventually expand the suite of human preclinical model systems, ranging from patient-specific cell lines to complex human embryonic tissues established from stem cells.