br Experimental Procedures br Author Contributions br Acknow

Experimental Procedures
Author Contributions
Acknowledgments
Introduction Human pluripotent stem acetylcholine inhibitors (PSCs) can differentiate into nearly all somatic cell types present in the human body and can generate clinically relevant numbers of cells for regenerative medicine. The advent of hiPSCs, derived from somatic cells by the exogenous expression of defined transcription factors, has overcome ethical issues associated with human embryonic stem cells (hESCs) and, when derived from the patient, may avoid immunological complications. Human iPSCs have also opened new avenues of research for the study of basic disease mechanisms and development of informative model systems for drug discovery. Although promising, significant limitations to the therapeutic use of hiPSCs remain unresolved. These include interline variations ranging from inconsistent transcription factor expression and differential DNA methylation to sporadic point mutations and chromosomal defects that affect in vitro differentiation, tumorigenicity, and potential clinical applications (Feng et al., 2010; Gore et al., 2011; Robinton and Daley, 2012). Moreover, current tests of hiPSC potency rely on extensive in vitro differentiation tests, in vivo teratoma assays in rodents (Maherali and Hochedlinger, 2008; Robinton and Daley, 2012) or bioinformatic and gene expression assays (Bock et al., 2011; Müller et al., 2011), which cannot be practically implemented into high-throughput hiPSC line generation designed to limit interline variability. The lack of suitable cell-surface marker panels and related affinity-based reagents for isolating high-quality hiPSCs and well-defined progeny significantly restricts our ability to minimize interline variability and employ hiPSCs for regenerative medicine. Although guidelines and animal-free methods have been proposed for the derivation and characterization of therapeutic and good manufacturing practice compliant hiPSCs (Buta et al., 2013; Funk et al., 2012; Maherali and Hochedlinger, 2008; Müller et al., 2010), no system is available to overcome safety and efficacy issues of hiPSCs analogous to immunophenotyping of blood lineages for identifying and isolating hematopoietic stem cells (HSCs). Although markers such as SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81 aid in the identification of hPSCs, few known surface markers and application-specific antibodies are restricted to the pluripotent state (Damjanov et al., 1982; Kannagi et al., 1983; Lowry et al., 2008). Moreover, as cell-surface proteins play critical roles in inter- and intracellular communication, a better understanding of the cell surface should inform the dynamic interplay between cells and their microenvironment that ultimately regulates how hPSCs interact with and respond to external cues and differentiate in a directed manner (Lian et al., 2013; Murry and Keller, 2008; Yan et al., 2005). Coupling this functional relevance with the fact that more than 60% of US Food and Drug Administration-approved drug therapies target membrane proteins, and 38% of disease-related proteins are membrane associated (Cheng et al., 2012; Yildirim et al., 2007), we aimed to generate a new resource derived from a targeted analytical approach, Cell Surface Capture (CSC) technology (Gundry et al., 2009, 2012; Hofmann et al., 2010; Wollscheid et al., 2009) that experimentally verifies extracellular domains of surface proteins. This resource, through its direct protein measurements, will foster the identification of proteins and epitopes useful for immunophenotyping and facilitate the identification of drugs that target hPSCs. The value of this resource is exemplified by the identification of proteins capable of marking live hPSCs and the identification of a small molecule inhibitor, STF-31, that allows for selective depletion of hPSCs from hESC and hiPSC-derived progeny.
Results