Biology subjects

Bayne, E. F.

Publications and source records attributed to Bayne, E. F..

5 recordsLinked to original sources

bioRxiv2026

Early multi-omic signatures and machine learning models predict cardiomyocyte differentiation efficiency and enable robust hPSC differentiation to cardiomyocytes

Protocols for generating cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) have existed for nearly two decades, yet manufacturing variability in terminal cell identity continues to limit clinical translation. To uncover the origin of fate divergence during hPSC-CM differentiation, we performed temporal transcriptomics, proteomics, and metabolomics of high and low efficiency differentiations. We identified significant early multi-omic divergence between differentiation batches and key pathways underlying fate divergence at critical differentiation stages included Wnt, MAPK, and glucose metabolism. Machine learning models trained on early candidate gene markers predicted hPSC-CM purity better than models using canonical cardiac development markers. Lastly, multi-omic insights informed perturbations, including Wnt and MAPK inhibition, which produced higher CM purities and yields. Our results showcase multi-omic analysis coupled with machine learning models as a powerful tool to identify cell fate determinants and enable robust manufacturing of complex cell products such as hPSC-derived cell therapies. TeaserMulti-omic analysis of hPSC-CM differentiation efficiency reveals early predictive features and enables robust differentiation.

cell biology↗

bioRxiv2023

High-throughput Extracellular Matrix Proteomics of Human Lungs Enabled by Photocleavable Surfactant and diaPASEF

The extracellular matrix (ECM) is a complex assembly of proteins that provide interstitial scaffolding and elastic recoil to human lungs. The pulmonary extracellular matrix (ECM) is increasingly recognized as an independent bioactive entity by creating biochemical and mechanical signals that influence disease pathogenesis, making it an attractive therapeutic target. However, the pulmonary ECM proteome ("matrisome") remains challenging to analyze by mass spectrometry due to its inherent biophysical properties and relatively low abundance. Here, we introduce a strategy designed for rapid and efficient characterization of the human pulmonary ECM using the photocleavable surfactant Azo. We coupled this approach with trapped ion mobility MS with diaPASEF to maximize depth of matrisome coverage. Using this strategy, we identify nearly 400 unique matrisome proteins with excellent reproducibility that are known to be important in lung biology, including key insoluble ECM proteins.

systems biology↗

bioRxiv2023

Lactate and Immunomagnetic-purified iPSC-derived Cardiomyocytes Generate Comparable Engineered Cardiac Tissue Constructs

Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely-used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared to magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACs-purified hiPSC-CMs impacts the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. After purification, hiPSC-CMs were combined with hiPSC-cardiac fibroblasts to create 3D hiPSC-ECT constructs maintained in culture for four weeks. There were no structural differences observed, and there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force, Ca2+ transients, and {beta}-adrenergic response revealed similar functional performance between purification methods. High-resolution mass spectrometry (MS)-based quantitative proteomics showed no significant difference in any protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable molecular and functional properties, and suggests lactate purification does not result in an irreversible change in hiPSC-CM phenotype. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/539642v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@54b370org.highwire.dtl.DTLVardef@d0bbc8org.highwire.dtl.DTLVardef@1d847e3org.highwire.dtl.DTLVardef@578c1b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Top-down Proteomics of Myosin Light Chain Isoforms Define Chamber-Specific Expression in the Human Heart

Myosin functions as the "molecular motor" of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an atrial and ventricular isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v, in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v and MLC-2a were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Overall, top-down proteomics allowed us an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

systems biology↗

bioRxiv2021

A Multi-omics Strategy Enabled by Sequential Metabolomics and Proteomics for Human Pluripotent Stem Cell-derived Cardiomyocytes

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show immense promise for patient-specific disease modeling, cardiotoxicity screening, and regenerative therapy development. However, hPSC-CMs in culture have not recapitulated the structural or functional properties of adult CMs in vivo thus far. To gain global insight into hPSC-CM biology, we established a multi-omics method for analyzing the hPSC-CM metabolome and proteome from the same cell culture, creating multi-dimensional profiles of hPSC-CMs. Specifically, we developed a sequential extraction to capture metabolites and proteins from the same hPSC-CM monolayer cultures, and analyzed these extracts using high-resolution mass spectrometry (MS). Using this method, we annotated 205 metabolites/lipids and 4,008 proteins from 106 cells with high reproducibility. We further integrated the proteome and metabolome measurements to create network profiles of molecular phenotypes for hPSC-CMs. Out of 310 pathways identified using metabolomics and proteomics, 40 pathways were considered significantly overrepresented (FDR-corrected p [≤] 0.05). Highly populated pathways included those involved in protein synthesis (ribosome, spliceosome), ATP generation (oxidative phosphorylation), and cardiac muscle contraction. This multi-omics method achieves deep coverage of metabolites and proteins, creating a multidimensional view of the hPSC-CM phenotype, which provides a strong technological foundation to advance the understanding of hPSC-CM biology.

systems biology↗