An important concern regarding stem cell

An important concern regarding stem cell based therapies is the potential for unwanted cell growth. In a previous study we showed that the TheraCyte encapsulation device is durable enough to withstand freezing without evidence of cell escape, suggesting that once THZ1 Hydrochloride Supplier are sealed in devices they will remain confined (Yakhnenko et al., 2012). Nevertheless, to more formally address this issue, BLI was used for spatial and temporal monitoring of encapsulated CyT49-luc cells in immunocompromised animals. Due to its sensitivity BLI is routinely employed for detection of micrometastases in animals with large primary tumors (Jenkins et al., 2003; Volk-Draper et al., 2012). Others have shown that as few as 3 cells could be detected in vivo and moreover, that 3 cells could be distinguished from 5 versus 10 versus 50 cells (Kim et al., 2010). Importantly, we did not find evidence of cell escape in any of 23 engrafted SCID/beige for up to 154days, the longest period evaluated. The data suggest that larger studies to extend these findings are warranted. Importantly, in the clinical setting of immunocompetent patients, if a cell escaped from the encapsulation device, it would presumably be recognized as foreign and destroyed. Nevertheless, an encapsulation device which prevents cell escape into the host is desirable as a safety measure and for retrievability of grafts.
Acknowledgments The authors thank Evert Kroon, Laura Martinson and Kevin D\'Amour at ViaCyte for generously providing CyT49 hES cells and for advice during the course of these studies. We also thank Nikunj Somia for the luciferase lentiviral vector. Funding for KK was provided by CIRM Bridges grant to California Polytechnic, TB1-01775. Funding for P. I-A was provided by CIRM and by The Hartwell Foundation. The funding sources played no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.
Introduction In human ES cells, introduction of stable genetic modifications by homologous recombination has been challenging for many laboratories. Most commonly, transgenes are introduced into ES cells via methods such as viral transduction or plasmid transfection. However, these methods lead to random integration within the genome (Tenzen et al., 2010). Although clones with stable transgene expression can be selected, most clones display transgene silencing upon ES cell differentiation. For example, even the very well characterized and strong beta globin promoter functions to appropriately induce transgene expression in only a minority of ES cell clones generated using a random integration strategy (Hatzistavrou et al., 2009). The use of zinc finger nucleases (ZFNs) is one way in which transgenes can be introduced at specific locations within the genome and potentially avoid gene silencing. ZFNs cause a site-specific DNA double strand break and enhance homologous recombination efficiency to introduce a construct of interest using gene targeting (Carroll, 2011). One method to prevent silencing of transgenic constructs is to select genomic loci of constitutively expressed genes for gene targeting. One such site in the human genome is the AAVS1 “safe harbor” locus which encodes the PPP1R12C gene and when targeted with transgenes results in stable gene expression (Dekelver et al., 2010; Hockemeyer et al., 2009). It has been shown that transgenic constructs targeted to the AAVS1 locus maintain expression in hematopoietic cells differentiated from human induced pluripotent stem (iPS) cells when using constitutive promoters (Garçon et al., 2013; Zou et al., 2011) or when using erythroid specific promoters (Chang and Bouhassira, 2012). Therefore, the AAVS1 locus offers a well-characterized site for transgene expression in human pluripotent stem cells. Here we demonstrate several technical advancements for using the AAVS1 locus to generate both reporter and knockdown constructs in human ES cells. We generate a reporter for hematopoietic cells and a knockdown construct that inhibits myeloid cell formation. One construct is designed to express a green fluorescent protein (GFP) reporter driven by a human CD43 promoter fragment that accurately reflects endogenous CD43 gene expression in hematopoietic cells. The second construct is designed to express short hairpin RNAs driven by the constitutive chicken actin (CA) promoter. By using hairpins that target PU.1, the master regulator of myeloid development, we demonstrate an efficient and stable gene knockdown that leads to a dramatic inhibition of myeloid development. Finally, we show that a simple lipid transfection method can generate a large number of correctly targeted clones much more efficiently and easily than previously published reports using electroporation (Hockemeyer et al., 2009). These techniques provide quick and reliable tools for expressing transgenes and developing reporter and knockdown lines to study hematopoiesis using human pluripotent stem cells.