Biology subjects

Palpant, N. J.

Publications and source records attributed to Palpant, N. J..

9 recordsLinked to original sources

bioRxiv2026

Protocol-dependent cardiomyocyte states determine disease modelling capacity of human iPSCs

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are widely used to model cardiovascular disease, yet numerous differentiation protocols generate cardiomyocytes with heterogeneous molecular and functional properties, complicating experimental design. Here we systematically compare sixteen commonly used cardiomyocyte differentiation protocols and characterize their resulting cell states using single-nucleus RNA sequencing, functional phenotyping and computational integration with human genetic data. Despite similar cardiomyocyte yields, protocols produced distinct transcriptional programs, subtype compositions and physiological properties. By integrating protocol-specific gene expression signatures with genome-wide association studies of cardiovascular traits, we identify cardiomyocyte states enriched for genetic architectures underlying specific diseases. These analyses accurately predict protocols most suitable for modelling particular disease contexts, including electrophysiological defects associated with Brugada syndrome and metabolic vulnerability relevant to myocardial infarction. Our results demonstrate that differentiation protocols encode biologically distinct cardiomyocyte states with differential disease relevance and establish a framework for aligning stem-cell differentiation strategies with human complex trait genetics to guide model selection. This approach enables rational design of iPSC-based disease models and highlights how population-scale genetic data can inform experimental systems in stem cell biology.

systems biology↗

bioRxiv2025

Contractile function maintains cardiomyocyte differentiation and inhibits cell cycle activity

Numerous endotherm species lose cardiac regenerative capacity shortly after birth, which is in contrast to many ectotherm species who regenerate throughout life. Whether the enhanced contractile function required for endothermy contributes to the cell-cycle exit remains to be explored. Herein, we use human cardiac organoids with advanced maturation combined with direct targeting of contraction using mavacamten and aficamten to enable exquisite control of active contraction over brief time windows. We show that transient inhibition of contraction re-activates the cell cycle. Multi-omics analyses demonstrated the cell cycle response to be mediated through a dedifferentiation-like process, which was swiftly reversed upon removal of the myosin inhibitors. Together these findings reveal that active contraction maintains differentiation including cell cycle arrest in cardiomyocytes.

cell biology↗

bioRxiv2025

Glycaemic variability underlies myocyte dysfunction and myocardial injury risk in diabetes

Heart disease is the leading cause of morbidity and mortality in individuals with diabetes, due largely to risks associated with ischaemic injuries such as myocardial infarction (MI). We use human population genetic data to demonstrate that current biomarkers of hyperglycaemia do not account for risk of post-MI mortality in diabetes patients. This study therefore systematically evaluates glycaemic stress underpinning cardiovascular risk in diabetes. Using in vivo and in vitro models, we demonstrate that glycaemic variability rather than hyperglycaemia alone is a dominant risk factor for heart muscle dysfunction and myocardial injury sensitivity in diabetes. These findings provide new preclinical models for mechanistic and drug discovery studies and inform strategies for managing cardiovascular outcomes in patients with diabetes.

cell biology↗

bioRxiv2023

High-content fluorescence bioassay to investigate pore formation, ion channel modulation and cell membrane lysis induced by venoms

Venoms comprise highly evolved bioactive molecules modulating ion channels, receptors, coagulation factors, and the cellular membrane. This array of targets and bioactivity requires high-content bioassays to aid the development of novel envenomation treatments and biotechnological and pharmacological agents. To address this gap in venoms research, we developed a fluorescence-based high-throughput and high-content cellular assay to simultaneously identify common cellular activities produced by venoms: membrane lysis, pore-formation, and ion channel modulation. By combining intracellular calcium with extracellular nucleic acid measurements, we distinguished these venom mechanisms using one cellular assay. We applied our high-content bioassay in three cell types exposed to venom components representing lytic, ion pore-forming or ion channel modulator toxins. Beyond the distinct profiles produced by these three types of action mechanisms, we found that the pore-forming latrotoxin -Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes, while the lytic bee peptide melittin is not selective. Furthermore, evaluation of snake venoms showed that Elapid species induced rapid membrane lysis, while Viper species showed variable to no activity on neuroblastoma cells. These findings demonstrate that our high-content bioassay distinguish clades and interspecific traits and capture the clinical observations at venom level and is capable of differentiate ion pore-forming from membrane lysis and ion channel modulation. We hope our research will accelerate the understanding of venom biology and the diversity of toxins inducing cytotoxic, cardiotoxic and neurotoxic effects and assist in identifying venom components whose properties could benefit humankind.

pharmacology and toxicology↗

bioRxiv2022

An integrated cell barcoding and computational analysis pipeline for scalable analysis of differentiation at single-cell resolution

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across multiple developmental lineage trajectories. In this study, we engineered barcoded iPSCs to generate an atlas of multilineage differentiation from pluripotency, encompassing a time-course of WNT-induced differentiation perturbed using modulators of WNT, BMP, and VEGF signalling. Computational mapping of in vitro cell types to in vivo developmental lineages revealed a diversity of iPSC-derived cell types comprising mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Coupling this atlas of in vitro differentiation with Summary data-based Mendelian Randomisation analysis of human complex traits, we identify the WNT-inhibitor protein TMEM88 as a putative regulator of mesendodermal cell types governing development of diverse cardiovascular and anthropometric traits. Using genetic loss of function models, we show that TMEM88 is required for differentiation of diverse endoderm and mesoderm cell lineages in vitro and that TMEM88 knockout in vivo results in a significant dysregulation of arterial blood pressure. This study provides an atlas of multilineage iPSC differentiation coupled with new molecular, computational, and statistical genetic tools to dissect genetic determinants of mammalian developmental physiology.

genomics↗

bioRxiv2022

Cellular Heterogeneity of Pluripotent Stem Cell Derived Cardiomyocyte Grafts is Mechanistically Linked to Treatable Arrhythmias

BackgroundExciting pre-clinical data have confirmed that human pluripotent stem cell derived cardiomyocytes (PSC-CMs) can remuscularise the injured or diseased heart, with several clinical trials now in planning or recruitment stages worldwide. However, ventricular arrhythmias are a predictable complication following engraftment of intramyocardially injected PSC-CMs. Therefore, there is an urgent unmet need to gain mechanistic insights and treatment strategies to control or prevent these engraftment arrhythmias (EAs). MethodsWe used a porcine model of myocardial infarction and PSC-CM transplantation to investigate efficacy of pharmacologic and catheter based anti-arrhythmic strategies in mitigating EAs. Furthermore, cell doses were robustly phenotyped using single cell ribonucleic acid sequencing and high parameter flow cytometry to identify cellular characteristics predictive of arrhythmogenesis. ResultsCombination therapy with amiodarone and ivabradine significantly reduced EA rate and burden following PSC-CM transplantation. Catheter ablation was also a feasible and effective treatment strategy which could be considered in the case of pharmacologically refractory arrhythmias. In addition, we show that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. We further describe two unique surface marker signatures, SIRPA+/CD90-/CD200+ and SIRPA+/CD90-/CD200-, which identify arrhythmogenic and non-arrhythmogenic cardiomyocytes respectively. ConclusionOur data deepens mechanistic understanding of EAs and suggests that modifications to current PSC-CM production and/or selection protocols could ameliorate this problem. We further show that current clinical pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias, an important safety consideration given several impending clinical trials.

cell biology↗

bioRxiv2022

Dynamic chromatin organization and regulatory interactions in human endothelial cell differentiation

BackgroundVascular endothelial cells are a mesoderm-derived lineage with many essential functions, including angiogenesis and coagulation. However, the gene regulatory mechanisms that underpin endothelial specialization are largely unknown, as are the roles of 3D chromatin organization in regulating endothelial cell transcription. MethodsTo investigate the relationships between 3D chromatin organization and gene expression in endothelial cell differentiation, we induced endothelial cell differentiation from human pluripotent stem cells and performed Hi-C and RNA-seq assays at specific timepoints in differentiation. ResultsOur analyses reveal that long-range intrachromosomal contacts increase over the course of endothelial cell differentiation, as do genomic compartment transitions between active and inactive states. These compartmental states are tightly associated with endothelial transcription. Dynamic topologically associating domain (TAD) boundaries strengthen and converge on an endothelial cell state, and nascent TAD boundaries are linked to the expression of genes that support endothelial cell specification. Relatedly, chromatin pairwise point interactions (DNA loops) increase in frequency during differentiation and are linked to the expression of genes with essential roles in vascular biology, including MECOM, TFPI, and KDR. To identify forms of regulation specific to endothelial cell differentiation, we compared the functional chromatin dynamics of endothelial cells with those of developing cardiomyocytes. Cardiomyocytes exhibit greater long-range cis interactions than endothelial cells, whereas endothelial cells have increased local intra-TAD interactions and much more abundant pairwise point interactions. ConclusionsGenome topology changes dynamically during endothelial differentiation, including acquisition of long-range cis interactions and new TAD boundaries, interconversion of hetero- and euchromatin, and formation of DNA loops. These chromatin dynamics guide transcription in the development of endothelial cells and promote the divergence of endothelial cells from related cell types such as cardiomyocytes.

developmental biology↗

bioRxiv2018

Evidence for minimal cardiogenic potential of Sca-1 positive cells in the adult mouse heart

BackgroundDespite modern pharmacotherapy, heart failure remains a major medical burden. The heart has a limited regenerative capacity, and bolstering regeneration might represent new therapeutic approaches for heart failure patients. Various progenitor cells in the heart have been proposed to have cardiomyogenic properties, but this evidence is based mostly on cell culture and transplantation studies. One population of interest is characterized by the expression of Stem Cell Antigen-1 (Sca-1). Here we tested the hypothesis that Sca-1+ cells are endogenous progenitors for cardiomyocytes in the adult heart.\n\nMethodsWe evaluated the innate cardiogenic potential of Sca-1+ cells in vivo by generating a novel mouse model to genetically lineage-trace the fate of Sca-1 expressing cells. This was accomplished by introducing a tamoxifen-inducible Cre-recombinase into the Sca-1 locus (Sca-1mCm/+). Crossing this mouse line to a Cre-dependent tdTomato reporter line allowed for genetic lineage-tracing of endogenous Sca-1+ cells (Sca-1mCmR26tdTomato). The frequency of Sca-1+ cardiomyocytes was quantified from dispersed cell preparations and confirmed by in situ histology.\n\nResultsWe validated the genetic lineage tracing mouse model in bone marrow and heart. Unlike previous publications suggesting significant cardiogenic potential, we found that less than 0.02% of cardiomyocytes per year were derived from Sca-1+ cells in the adult heart under homeostatic conditions. At six months after myocardial infarction, we found less than 0.01% of cardiomyocytes were derived from Sca-1+ cells.\n\nConclusionOur results show that Sca-1+ cells in the adult heart have minimal cardiogenic potential under homeostatic conditions or in response to myocardial infarction.

cell biology↗

bioRxiv2016

Isolator: accurate and stable analysis of isoform-level expression in RNA-Seq experiments

While RNA-Seq has enabled great progress towards the goal of wide-scale isoform-level mRNA quantification, short reads have limitations when resolving complex or similar sets of isoforms. As a result, estimates of isoform abundance carry far more uncertainty than those made at the gene level. When confronted with this uncertainty, commonly used methods produce estimates that are often high-variance--small perturbations in the data often produce dramatically different results, confounding downstream analysis. We introduce a new method, Isolator, which analyzes all samples in an experiment in unison using a simple Bayesian hierarchical model. Combined with aggressive bias correction, it produces estimates that are simultaneously accurate and show high agreement between samples. In a comprehensive comparison of accuracy and variance, we show that this property is unique to Isolator. We further demonstrate that the approach of modeling an entire experiment enables new analyses, which we demonstrate by examining splicing monotonicity across several time points in the development of human cardiomyocyte cells.

bioinformatics↗