Embryonic Stem Cell-Derived Cardiomyocyte Heterogeneity and the Isolation of Immature and Committed Cells for Cardiac Remodeling and Regeneration

Pluripotent stem cells represent one promising source for cell replacement therapy in heart, but differentiating embryonic stem cell-derived cardiomyocytes (ESC-CMs) are highly heterogeneous and show a variety of maturation states. In this study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter-driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass, and hypoxia-resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction

Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping

Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of N-glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.</p

Heart Disease, Clinical Proteomics and Mass Spectrometry

Heart disease is the leading cause of mortality and morbidity in the world. As such, biomarkers are needed for the diagnosis, prognosis, therapeutic monitoring and risk stratification of acute injury (acute myocardial infarction (AMI)) and chronic disease (heart failure). The procedure for biomarker development involves the discovery, validation, and translation into clinical practice of a panel of candidate proteins to monitor risk of heart disease. Two types of biomarkers are possible; heart-specific and cardiovascular pulmonary system monitoring markers. Here we review the use of MS in the process of cardiac biomarker discovery and validation by proteomic analysis of cardiac myocytes/tissue or serum/plasma. An example of the use of MS in biomarker discovery is given in which the albumin binding protein sub-proteome was examined using MALDI-TOF MS/MS. Additionally, an example of MS in protein validation is given using affinity surface enhanced laser desorption ionization (SELDI) to monitor the disease-induced post-translational modification and the ternary status of myoctye-originating protein, cardiac troponin I in serum

Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes

When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells

Plasma Metabolome Normalization in Rheumatoid Arthritis Following Initiation of Methotrexate and the Identification of Metabolic Biomarkers of Efficacy

Methotrexate (MTX) efficacy in the treatment of rheumatoid arthritis (RA) is variable and unpredictable, resulting in a need to identify biomarkers to guide drug therapy. This study evaluates changes in the plasma metabolome associated with response to MTX in RA with the goal of understanding the metabolic basis for MTX efficacy towards the identification of potential metabolic biomarkers of MTX response. Plasma samples were collected from healthy control subjects (n = 20), and RA patients initiating MTX therapy (n = 20, 15 mg/week) before and after 16 weeks of treatment. The samples were analyzed by a semi-targeted metabolomic analysis, and then analyzed by univariate and multivariate methods, as well as an enrichment analysis. An MTX response was defined as a clinically significant reduction in the disease activity score in 28 joints (DAS-28) of greater than 1.2; achievement of clinical remission, defined as a DAS-28 p = 1.1 × 10−16), fatty acids (p = 8.0 × 10−12), and ceramides (p = 9.8 × 10−13). Stratification based on responses to MTX identified various metabolites differentially impacted in responders and non-responders including glucosylceramides (GlcCer), phosphatidylcholines (PC), sphingomyelins (SM), phosphatidylethanolamines (PE), choline, inosine, hypoxanthine, guanosine, nicotinamide, and itaconic acid (p < 0.05). In conclusion, RA is associated with significant alterations to the plasma metabolome displaying at least partial normalization following 16 weeks of MTX therapy. Changes in multiple metabolites were found to be associated with MTX efficacy, including metabolites involved in fatty acid/lipid, nucleotide, and energy metabolism

Veneer Is a Webtool for Rapid, Standardized, and Transparent Interpretation, Annotation, and Reporting of Mammalian Cell Surface <i>N</i>‑Glycocapture Data

Currently, no consensus exists regarding criteria required to designate a protein within a proteomic data set as a cell surface protein. Most published proteomic studies rely on varied ontology annotations or computational predictions instead of experimental evidence when attributing protein localization. Consequently, standardized approaches for analyzing and reporting cell surface proteome data sets would increase confidence in localization claims and promote data use by other researchers. Recently, we developed Veneer, a web-based bioinformatic tool that analyzes results from cell surface N-glycocapture workflowsthe most popular cell surface proteomics method used to date that generates experimental evidence of subcellular location. Veneer assigns protein localization based on defined experimental and bioinformatic evidence. In this study, we updated the criteria and process for assigning protein localization and added new functionality to Veneer. Results of Veneer analysis of 587 cell surface N-glycocapture data sets from 32 published studies demonstrate the importance of applying defined criteria when analyzing cell surface proteomics data sets and exemplify how Veneer can be used to assess experimental quality and facilitate data extraction for informing future biological studies and annotating public repositories

Veneer Is a Webtool for Rapid, Standardized, and Transparent Interpretation, Annotation, and Reporting of Mammalian Cell Surface <i>N</i>‑Glycocapture Data

Currently, no consensus exists regarding criteria required to designate a protein within a proteomic data set as a cell surface protein. Most published proteomic studies rely on varied ontology annotations or computational predictions instead of experimental evidence when attributing protein localization. Consequently, standardized approaches for analyzing and reporting cell surface proteome data sets would increase confidence in localization claims and promote data use by other researchers. Recently, we developed Veneer, a web-based bioinformatic tool that analyzes results from cell surface N-glycocapture workflowsthe most popular cell surface proteomics method used to date that generates experimental evidence of subcellular location. Veneer assigns protein localization based on defined experimental and bioinformatic evidence. In this study, we updated the criteria and process for assigning protein localization and added new functionality to Veneer. Results of Veneer analysis of 587 cell surface N-glycocapture data sets from 32 published studies demonstrate the importance of applying defined criteria when analyzing cell surface proteomics data sets and exemplify how Veneer can be used to assess experimental quality and facilitate data extraction for informing future biological studies and annotating public repositories

Are These Cardiomyocytes? Protocol Development Reveals Impact of Sample Preparation on the Accuracy of Identifying Cardiomyocytes by Flow Cytometry

Summary: Several protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation. : Waas and colleagues demonstrate pitfalls with popular antibodies and sample preparation conditions commonly used for the assessment of cardiomyocyte identity within differentiation cultures. By using a rigorous fit-for-purpose workflow, the authors developed and validated a comprehensive protocol to accurately assess cardiomyocyte identity within hPSC-CM cultures. The new protocol includes stepwise instructions to facilitate its implementation by experts and novices alike. Keywords: quality control, flow cytometry, troponin, cardiomyocytes, mass spectrometry, standard operating protoco