Coby B. Carlson

Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

Phenotypic Screening with Human iPS Cell–Derived Cardiomyocytes HTS-Compatible Assays for Interrogating Cardiac Hypertrophy

A major hurdle for cardiovascular disease researchers has been the lack of robust and physiologically relevant cell-based assays for drug discovery. Derivation of cardiomyocytes from human-induced pluripotent stem (iPS) cells at high purity, quality, and quantity enables the development of relevant models of human cardiac disease with source material that meets the demands of high-throughput screening (HTS). Here we demonstrate the utility of iPS cell–derived cardiomyocytes as an in vitro model of cardiac hypertrophy. Exposure of cardiomyocytes to endothelin 1 (ET-1) leads to reactivation of fetal genes, increased cell size, and robust expression of B-type natriuretic peptide (BNP). Using this system, we developed a suite of assays focused on BNP detection, most notably a high-content imaging-based assay designed for phenotypic screening. Miniaturization of this assay to a 384-well format enabled the profiling of a small set of tool compounds known to modulate the hypertrophic response. The assays described here provide consistent and reliable results and have the potential to increase our understanding of the many mechanisms underlying this complex cardiac condition. Moreover, the HTS-compatible workflow allows for the incorporation of human biology into early phases of drug discovery and development.

Solid-phase synthesis of acridine-based threading intercalator peptides

The preparation of a novel acridine-based amino acid is reported. This N-Alloc-protected monomer can be coupled and deprotected under solid-phase peptide synthesis procedures to create acridine peptide conjugates as potential threading intercalators. A peptide containing this novel amino acid undergoes spectral changes in the presence of duplex DNA and RNA consistent with intercalative binding.

Inhibitory properties of nucleic acid-binding ligands on protein synthesis

The use of small molecule inhibitors in the study of cellular processes is a powerful approach to understanding gene function. During the course of a high throughput screen for novel inhibitors of eukaryotic translation, we identified a number of nucleic acid binding ligands that showed activity in our assay. When tested on a panel of mRNA transcripts displaying different modes of translation initiation, these ligands showed a range of biological activities – with some inhibiting both cap‐dependent and internal initiation and others preferentially blocking internal initiation. We used this information to identify a novel threading intercalator that inhibits Hepatitis C virus internal initiation.

Selection of small-molecule mediators of the RNA regulation of PKR, the RNA-dependent protein kinase

The RNA‐dependent protein kinase (PKR) is a component of the interferon antiviral response and a member of the class of RNA‐binding proteins with a double‐stranded RNA binding motif. PKR is activated when it binds to double‐stranded RNA (dsRNA) or viral replicative intermediates that comprise dsRNA and this activation results in the inhibition of protein synthesis. Some viruses circumvent this activity through the synthesis of highly structured decoy RNAs that bind PKR and block activation. Small‐molecule mediators of the binding of PKR to these RNA inhibitors would be useful tools to further define the importance of specific PKR–RNA complexes in vivo and may possess antiviral activity. Here we investigate the ability of a library of structurally diverse peptide–acridine conjugates (PACs) to target a complex formed between the dsRNA binding domain (dsRBD) of PKR and a viral RNA inhibitor. We used a novel screening method based on the cleavage of RNA ligands with ethylenediaminetetraacetic acid⋅Fe modified protein. The selection revealed a PAC (9‐anilinoacridine‐4‐Hyp‐Nap‐Nap, where Hyp is trans‐4‐hydroxyproline and Nap is 1‐napthylalanine), able to inhibit the binding of the PKR dsRBD to RNA with an IC50 value of 10±5 μM. Furthermore, the structural requirements for inhibition by the selected PAC were substantiated in an independent PKR activation assay. We found that the potency of inhibition by an intercalating ligand can be increased by the introduction of a substituent that does not increase the overall charge of the molecule. This result is important for the design of inhibitors of PKR–RNA binding that function inside living cells.

Human iPS Cell-Derived Cardiomyocytes Carrying MHC-R403Q Exhibit Aspects of Hypertrophic Cardiomyopathy in vitro

Hypertrophic cardiomyopathy (HCM) is an inherited disease of the heart muscle that affects approximately 1 in 500 individuals. The condition is characterized by enlarged cardiac myocytes, thickened ventricular walls, non-compliant muscle structure, changes in blood pressure, electrical arrhythmias, and shortness of breath. The Arg-to-Gln switch at amino acid 403 of beta-myosin heavy chain (MHC-R403Q) is the most prevalent mutation associated with familial HCM. Human induced pluripotent stem (iPS) cell-derived cardiomyocytes can be generated in highly pure and large quantities from both control and affected individuals to provide a path forward toward better mechanistic understanding of complex heart diseases.Endothelin-1 (ET-1)-induced cardiac hypertrophy in human iPS cell-derived cardiomyocytes exhibits several classic hallmarks of cardiac hypertrophy including up-regulation of the fetal gene expression program, cytoskeletal rearrangements, and an increase in cardiomyocyte cell size (Carlson et al., 2013). Similar to the induced hypertrophic state, here we describe novel information regarding innate cardiac hypertrophy associated with MHC-R403Q. iPS cell-derived cardiomyocytes were generated from an MHC-R403Q-positive individual exhibiting the HCM phenotype. MHC-R403Q cardiomyocytes were generated at high purity (>95% TNNT-positive), exhibited the expected cardiac morphology, and showed autonomous contractile activity similar to the control cardiomyocytes (non-MHC-R403Q). Transcript profiling demonstrated that basal gene expression of MHC-R403Q cardiomyocytes was similar to control cardiomyocytes that had been induced with ET-1 into a hypertrophic state. Similarly, basal BNP levels in MHC-R403Q cardiomyocytes were higher than those of uninduced control cardiomyocytes. Interestingly, when treated with ET-1, cardiomyocytes from both backgrounds exhibited increases in BNP expression to similar levels. These data suggest that MHC-R403Q cardiomyocytes may have an innate predisposition toward familial hypertrophic cardiomyopathy and underscore the advantages of modeling cardiovascular disease through the use of iPSC technology.

Abstract 27: Label-free, real-time analysis of endothelial cell morphogenesis using iPSC-derived endothelial cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Endothelial cell (EC) morphogenesis during the early stages of angiogenesis is a highly dynamic process tightly regulated by growth factor activity in the extracellular milieu. Other in vitro models for the analysis of EC morphogenesis have previously been developed to understand the cellular processes and the impact of soluble cues and pharmacological inhibitors. However, these assays typically utilize end-point analysis of primary endothelial cells (eg. HUVEC), which are a physiologically relevant vascular cell type but do exhibit variability from the diversity of donors. Additionally, current methods for measuring morphogenesis (eg. proliferation and migration) only capture a snapshot of cell function owing to potentially destructive labeling of the cells and do not capture the complexity of cellular processes involved in EC morphogenesis Here, we describe use of a real-time system for monitoring of cellular processes using electronic cell sensor array technology. The cellular model tested on this impedance-based platform was a well-defined human induced pluripotent stem cell (iPSC)-derived endothelial cells. Following assay optimization and workflow improvement, we assessed the impact of serum, growth factors (e.g. VEGF, EGF, FGF-2), and small-molecule angiogenesis inhibitors (e.g. Sunitinib, SU1498) on the proliferation, migration, and invasion of iPSC-derived ECs compared to primary cells. We observed that real-time monitoring of such cellular processes offers distinct and important advantages over traditional end-point assays - specifically, the ability to measure receptor activation and quantify morphologic and adhesive remodeling of EC upon growth factor activation or signaling inhibition. By better understanding cellular morphogenesis in vitro, we can generate a picture of what soluble cues regulate the process and how pharmacological agents can modulate the different cell fates underlying morphogenesis. The use of iPSC-derived endothelial cells provides a robust and reproducible source of cells that perform equivalently to the standard that is HUVEC. The combination of iPSC-derived EC together with a real time monitoring system provides a biologically relevant human model system. Citation Format: David Mann, David Belair, Coby Carlson, Arne Thompson, Yama Abassi, Jeff Irelan. Label-free, real-time analysis of endothelial cell morphogenesis using iPSC-derived endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 27. doi:10.1158/1538-7445.AM2014-27