Kenneth R. Boheler

A Mass Spectrometric-Derived Cell Surface Protein Atlas

Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

Physical developmental cues for the maturation of human pluripotent stem cell-derived cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are the most promising source of cardiomyocytes (CMs) for experimental and clinical applications, but their use is largely limited by a structurally and functionally immature phenotype that most closely resembles embryonic or fetal heart cells. The application of physical stimuli to influence hPSC-CMs through mechanical and bioelectrical transduction offers a powerful strategy for promoting more developmentally mature CMs. Here we summarize the major events associated with in vivo heart maturation and structural development. We then review the developmental state of in vitro derived hPSC-CMs, while focusing on physical (electrical and mechanical) stimuli and contributory (metabolic and hypertrophic) factors that are actively involved in structural and functional adaptations of hPSC-CMs. Finally, we highlight areas for possible future investigation that should provide a better understanding of how physical stimuli may promote in vitro development and lead to mechanistic insights. Advances in the use of physical stimuli to promote developmental maturation will be required to overcome current limitations and significantly advance research of hPSC-CMs for cardiac disease modeling, in vitro drug screening, cardiotoxicity analysis and therapeutic applications.

Developmental cues for the maturation of metabolic, electrophysiological and calcium handling properties of human pluripotent stem cell-derived cardiomyocytes

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, are abundant sources of cardiomyocytes (CMs) for cell replacement therapy and other applications such as disease modeling, drug discovery and cardiotoxicity screening. However, hPSC-derived CMs display immature structural, electrophysiological, calcium-handling and metabolic properties. Here, we review various biological as well as physical and topographical cues that are known to associate with the development of native CMs in vivo to gain insights into the development of strategies for facilitated maturation of hPSC-CMs.

Human pluripotent stem cell-derived cardiomyocytes for heart regeneration, drug discovery and disease modeling: from the genetic, epigenetic, and tissue modeling perspectives

Heart diseases remain a major cause of mortality and morbidity worldwide. However, terminally differentiated human adult cardiomyocytes (CMs) possess a very limited innate ability to regenerate. Directed differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into CMs has enabled clinicians and researchers to pursue the novel therapeutic paradigm of cell-based cardiac regeneration. In addition to tissue engineering and transplantation studies, the need for functional CMs has also prompted researchers to explore molecular pathways and develop strategies to improve the quality, purity and quantity of hESC-derived and iPSC-derived CMs. In this review, we describe various approaches in directed CM differentiation and driven maturation, and discuss potential limitations associated with hESCs and iPSCs, with an emphasis on the role of epigenetic regulation and chromatin remodeling, in the context of the potential and challenges of using hESC-CMs and iPSC-CMs for drug discovery and toxicity screening, disease modeling, and clinical applications.

PTHGRN: unraveling post-translational hierarchical gene regulatory networks using PPI, ChIP-seq and gene expression data

Interactions among transcriptional factors (TFs), cofactors and other proteins or enzymes can affect transcriptional regulatory capabilities of eukaryotic organisms. Post-translational modifications (PTMs) cooperate with TFs and epigenetic alterations to constitute a hierarchical complexity in transcriptional gene regulation. While clearly implicated in biological processes, our understanding of these complex regulatory mechanisms is still limited and incomplete. Various online software have been proposed for uncovering transcriptional and epigenetic regulatory networks, however, there is a lack of effective web-based software capable of constructing underlying interactive organizations between post-translational and transcriptional regulatory components. Here, we present an open web server, post-translational hierarchical gene regulatory network (PTHGRN) to unravel relationships among PTMs, TFs, epigenetic modifications and gene expression. PTHGRN utilizes a graphical Gaussian model with partial least squares regression-based methodology, and is able to integrate protein–protein interactions, ChIP-seq and gene expression data and to capture essential regulation features behind high-throughput data. The server provides an integrative platform for users to analyze ready-to-use public high-throughput Omics resources or upload their own data for systems biology study. Users can choose various parameters in the method, build network topologies of interests and dissect their associations with biological functions. Application of the software to stem cell and breast cancer demonstrates that it is an effective tool for understanding regulatory mechanisms in biological complex systems. PTHGRN web server is publically available at web site http://www.byanbioinfo.org/pthgrn.

Inhibition of an NAD+ Salvage Pathway Provides Efficient and Selective Toxicity to Human Pluripotent Stem Cells

The tumorigenic potential of human pluripotent stem cells (hPSCs) is a major limitation to the widespread use of hPSC derivatives in the clinic. Here, we demonstrate that the small molecule STF‐31 is effective at eliminating undifferentiated hPSCs across a broad range of cell culture conditions with important advantages over previously described methods that target metabolic processes. Although STF‐31 was originally described as an inhibitor of glucose transporter 1, these data support the reclassification of STF‐31 as a specific NAD+ salvage pathway inhibitor through the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). These findings demonstrate the importance of an NAD+ salvage pathway in hPSC biology and describe how inhibition of NAMPT can effectively eliminate hPSCs from culture. These results will advance and accelerate the development of safe, clinically relevant hPSC‐derived cell‐based therapies.

Nitric Oxide‐cGMP‐PKG Pathway Acts on Orai1 to Inhibit the Hypertrophy of Human Embryonic Stem Cell‐Derived Cardiomyocytes

Cardiac hypertrophy is an abnormal enlargement of heart muscle. It frequently results in congestive heart failure, which is a leading cause of human death. Previous studies demonstrated that the nitric oxide (NO), cyclic GMP (cGMP), and protein kinase G (PKG) signaling pathway can inhibit cardiac hypertrophy and thus improve cardiac function. However, the underlying mechanisms are not fully understood. Here, based on the human embryonic stem cell‐derived cardiomyocyte (hESC‐CM) model system, we showed that Orai1, the pore‐forming subunit of store‐operated Ca2+ entry (SOCE), is the downstream effector of PKG. Treatment of hESC‐CMs with an α‐adrenoceptor agonist phenylephrine (PE) caused a marked hypertrophy, which was accompanied by an upregulation of Orai1. Moreover, suppression of Orai1 expression/activity using Orai1‐siRNAs or a dominant‐negative construct Orai1G98A inhibited the hypertrophy, suggesting that Orai1‐mediated SOCE is indispensable for the PE‐induced hypertrophy of hESC‐CMs. In addition, the hypertrophy was inhibited by NO and cGMP via activating PKG. Importantly, substitution of Ala for Ser34 in Orai1 abolished the antihypertrophic effects of NO, cGMP, and PKG. Furthermore, PKG could directly phosphorylate Orai1 at Ser34 and thus prevent Orai1‐mediated SOCE. Together, we conclude that NO, cGMP, and PKG inhibit the hypertrophy of hESC‐CMs via PKG‐mediated phosphorylation on Orai1‐Ser‐34. These results provide novel mechanistic insights into the action of cGMP‐PKG‐related antihypertrophic agents, such as NO donors and sildenafil. Stem Cells 2015;33:2973–2984

Proteomic Analysis of Human Pluripotent Stem Cell-Derived, Fetal, and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and Maturation

Background—Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs), but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches, but the global proteome has not been examined. Furthermore, most hESC-CM studies focus on pathways important for cardiac differentiation, rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs, hESC-derived VCMs, human fetal and human adult ventricular and atrial CMs. Methods and Results—Using two-dimensional–differential-in-gel electrophoresis, 121 differentially expressed (>1.5-fold; P<0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator–activated receptor &agr; signaling in cardiac maturation. Consistently, WY-14643, a peroxisome proliferator–activated receptor &agr; agonist, increased fatty oxidative enzyme level, hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line, treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold, signifying their maturation. Conclusions—We conclude that the peroxisome proliferator–activated receptor &agr; and thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs.

PlaMoM: a comprehensive database compiles plant mobile macromolecules.

In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein–protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/.

N-glycoprotein surfaceomes of four developmentally distinct mouse cell types

Detailed knowledge of cell surface proteins present during early embryonic development remains limited for most cell lineages. Due to the relevance of cell surface proteins in their functional roles controlling cell signaling and their utility as accessible, nongenetic markers for cell identification and sorting, the goal of this study was to provide new information regarding the cell surface proteins present during early mouse embryonic development.