Biology subjects

Sebra, R.

Publications and source records attributed to Sebra, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Critical assessment of intratumor and low-biomass microbiome using long-read sequencing

The detection of low-biomass microbial DNA in human tissues is often confounded by contamination, as demonstrated in the debates over the existence of microbiomes in the placenta, brain, blood, and tumors. Here we show that genomic DNA fragment length serves as a discriminator: while genuine microbiome genomes have long genomic DNA fragments, contaminant DNA is typically short. Using germ-free mouse tissues with bacterial spike-ins and human cell lines, we developed a metric that normalizes microbial read length to host read length. Across multiple human tumor and normal tissues, we found genuine microbiome signals are largely limited to tissues with natural microbial exposure (e.g., gastrointestinal tract, cervix, vagina, skin), while other tissues (e.g. kidney, brain, blood, and placenta) showed no evidence of resident microbiome. These findings support DNA fragment length as a metric for quality controlling low-biomass microbiome profiling, clarifying the debates and strengthen future studies of resident microbiome in tissues.

genomics↗

bioRxiv2026

Dysregulated TGFb-ERK Signaling Drives Aberrant Extracellular Matrix Production in Noonan Syndrome-Associated Pulmonary Valve Stenosis

Pulmonary valve stenosis (PVS) is the most common congenital heart defect in Noonan syndrome (NS) and related RASopathies, yet the molecular mechanisms linking pathogenic variants to the valve pathology remain poorly defined. Here, we utilized a human iPSC-based valve differentiation platform to generate the cardiac valve cell lineages--including fibrosa and spongiosa valve interstitial cell (VIC) subtypes. CRISPR-edited iPSCs harboring NS gain-of-function RAS/MAPK and Noonan syndrome with multiple lentigines (NSML) dominant-negative RAS/MAPK variants exhibited early defects in mesodermal and endocardial specification in all genotypes. Additionally, NS-iPSC endocardial cells exhibited defects in endothelial-to-mesenchymal transition (EndMT) specifically towards fibrosa VICs, which was most pronounced in PTPN11N308D (N308D) cells. Single-cell transcriptomics revealed widespread dysregulation of extracellular matrix (ECM) programs in N308D fibrosa VICs, including increased expression of collagens and proteoglycans, as well as dysregulation of multiple genes involved in ECM remodeling. We also detected activation of RAS-MAPK, TGF{beta}, and fibrosis-associated pathways in our transcriptional dataset. Mass spectrometry-based phosphoproteomics confirmed coordinated increases in ERK, PKC, and stress-related kinases, as well as enhanced activity of the TGF{beta} receptor. Functionally, N308D fibrosa VICs exhibited exaggerated upregulation of ECM genes in the presence of TGF{beta}2 ligand, suggesting that these cells are hypersensitive to TGF{beta} stimulation. Furthermore, we demonstrated that this pathological ECM-program occurs independently of BAMBI, a negative regulator of TGF{beta} signaling that was found to be decreased in N308D fibrosa VICs. Lastly, we performed histopathological analyses of stenotic pulmonary valves from two NS infants, which demonstrated marked overproduction and disorganization of ECM, mirroring the findings from our iPSC-based disease model. Together, our data reveal a central mechanism where NS-associated alleles sensitize fibrosa VICs to TGF{beta}, which leads to aberrant downstream signaling and drives the pathological ECM program in NS-associated PVS.

cell biology↗

bioRxiv2025

Cell-type-resolved NRXN1 isoforms across human brain tissues and hiPSC organoids

NRXN1 undergoes extensive alternative splicing that generates a highly diverse repertoire of isoforms, diversifying protein-protein interactions, shaping synaptic specialization, and contributing to neuropsychiatric disease when disrupted. However, the splicing landscape of NRXN1 across distinct human brain cell types remains poorly defined. Because NRXN1 expression level is relatively low in adult brains and human induced pluripotent stem cell (hiPSC) derived neurons, single-cell long-read cDNA sequencing often provides insufficient coverage to capture its full isoform diversity, particularly across heterogeneous cell populations. To address this gap, we developed an integrative sequencing strategy that combines single-cell transcriptomics, targeted enrichment of NRXN1 transcripts, and long-read sequencing. This approach reveals a comprehensive catalog of cell-type resolved NRXN1 isoforms across adult and fetal human postmortem brains as well as hiPSC-derived cortical organoids. From the adult prefrontal cortex (PFC) region, our analyses reveal distinct splicing programs across interneuron subtypes, pyramidal neurons, and glial lineages. Comparisons between prenatal and adult brains indicate that NRXN1 isoform profiles are established during early development and remain stable throughout neuronal maturation. In hiPSC organoid models, splicing profiles partially mirror both developmental and mature brain patterns, with a subset of splice sites exhibiting cell type - and stage-specific divergence. In the cerebellum of an autism case and in organoids derived from schizophrenia patients, carrying non-recurrent heterozygous NRXN1 deletions, we identify disrupted isoform expression and mutant NRXN1 isoforms and characterize their distribution across diverse cell types. Using this integrated long-read sequencing framework in patient-derived organoids, we further show that antisense oligonucleotide (ASO) targeting of mutant splice junctions reduces gain-of-function NRXN1 isoforms and reshapes splicing patterns in a cell type-specific manner, providing a platform for evaluating ASO efficiency. Together, these findings help understand the cell type-specific splicing landscape of NRXN1 and establish a framework for decoding cell type-specific isoform diversity and assessing transcript-targeted therapies for genes with low expression levels.

neuroscience↗

bioRxiv2025

Development and validation of HIV SMRTcap for the characterization of HIV-1 reservoirs across tissues and subtypes

Human Immunodeficiency Virus type 1 (HIV-1) is responsible for the global HIV/AIDS epidemic and the establishment of an integrated HIV-1 reservoir remains the primary obstacle to cure. Upon therapy interruption, reactivation of the persistent HIV-1 reservoir propagates viral rebound and mediates continued immunological decline. While furthering understanding of the HIV-1 reservoir is essential for HIV-1 cure, commonly used sequencing strategies are often limited by the reliance on short-read sequencing across separate assays to determine integration sites and proviral integrity - something that does not always adequately resolve complex human genomic repeats or low complexity regions. Simultaneous identification of proviral integration sites and proviral integrity at the single molecule level would enable HIV-1 reservoir characterization with minimal imputation or bioinformatic reconstruction. Here we present HIV Single Molecule Real Time Capture (HIV SMRTcap), a novel molecular and computational pipeline that directly and simultaneously identifies HIV-1 integration sites, defines proviral integrity, and characterizes clonal expansion of HIV-1 provirus-containing cells with single molecule resolution. In combination with long-read, single-molecule, real-time (SMRT) sequencing and custom analytic pipelines, HIV SMRTcap enables a highly comprehensive characterization of HIV-1 reservoirs. Moreover, we demonstrate here that HIV SMRTcap performs robustly across the major global subtypes (HIV-1 subtype A, B, C, D and A/D recombinant viruses), and can use both cell- and tissue-derived inputs, including samples from antiretroviral therapy (ART) treated individuals with undetectable viral loads. Our results demonstrate that HIV SMRTcap serves as a comprehensive, robust method for unbiased HIV-1 reservoir characterization. Used alone, or in combination with single-cell based methods, HIV SMRTcap will enable novel exploration of viral reservoirs across subtypes and in tissue-specific compartments, providing critical information needed to inform HIV-1 cure.

genomics↗

bioRxiv2025

Regulation of airway fumarate by host and pathogen promotes S. aureus pneumonia

Staphylococcus aureus is a leading cause of healthcare-associated pneumonia, contributing significantly to morbidity and mortality worldwide. As a ubiquitous colonizer of the upper respiratory tract, S. aureus must undergo substantial metabolic adaptation to achieve persistent infection in the distinctive microenvironment of the lung. We observed that fumC, which encodes the enzyme that converts fumarate to malate, is highly conserved with low mutation rates in S. aureus isolates from chronic lung infections. Fumarate, a pro-inflammatory metabolite produced by macrophages during infection, is regulated by the host fumarate hydratase (FH) to limit inflammation. Here, we demonstrate that fumarate, which accumulates in the chronically infected lung, is detrimental to S. aureus, blocking primary metabolic pathways such as glycolysis and oxidative phosphorylation (OXPHOS). This creates a metabolic bottleneck that drives staphylococcal FH (FumC) activity for airway adaptation. FumC not only degrades fumarate but also directs its utilization into critical pathways including the tricarboxylic acid (TCA) cycle, gluconeogenesis and hexosamine synthesis to maintain metabolic fitness and form a protective biofilm. Itaconate, another abundant immunometabolite in the infected airway enhances FumC activity, in synergy with fumarate. In a mouse model of pneumonia, a {Delta}fumC mutant displays significant attenuation compared to its parent and complemented strains, particularly in fumarate- and itaconate-replete conditions. Our findings underscore the pivotal role of immunometabolites in promoting S. aureus pulmonary adaptation.

microbiology↗

bioRxiv2025

The human immunoglobulin heavy chain constant gene locus is enriched for large complex structural variants and coding polymorphisms that vary in frequency among human populations

The human immunoglobulin heavy chain constant (IGHC) domain of antibodies (Ab) is responsible for effector functions critical to immunity. This domain is encoded by genes in the IGHC locus, where descriptions of genomic diversity remain incomplete. We utilized long-read sequencing to build an IGHC haplotype/variant catalog from 105 individuals of diverse ancestry. We discovered uncharacterized single nucleotide variants (SNV) and large structural variants (SVs, n=7), representing new genes and alleles enriched for non-synonymous substitutions, highlighting potential functional effects. Of the 221 identified IGHC alleles, 192 were novel. SNV, SV, and gene allele/genotype frequencies revealed population differentiation, including (i) hundreds of SNVs in African and East Asian populations exceeding a fixation index (FST) of 0.3, and (ii) an IGHG4 haplotype carrying coding variants uniquely enriched in Asian populations. Our results illuminate missing signatures of IGHC diversity and establish a new foundation for investigating IGHC germline variation in Ab function and disease.

genomics↗

bioRxiv2024

Mapping of Multilineage Tumor Cell Populations in Mouse Bladder Cancer

In bladder cancer (BLCA), the cellular expression of lineage markers with predictive biomarker function can have important implications for tumor progression, treatment response, and survival. However, tumor heterogeneity and the coexistence of distinct tumor subpopulations can complicate the utility of using a primary, clinically assigned tumor signature. In this report, we have applied the reference atlas, Tabula Sapiens, and deep learning model, UniCell, to conduct in-depth transcriptional analysis of unique lineage marker-defined cell clusters in a carcinogen (BBN) induced bladder cancer model. UniCell deconvolution has identified tumor populations, including urothelial, adenocarcinoma, squamous cell carcinoma, and mesenchymal tumor populations, each with cell-intrinsic gene expression signatures relevant to human BLCA progression. The identified tumor clusters contain uniquely basal, luminal, stromal, or hybrid cells in cell lineage marker expression. To understand the significance of these populations during progression, we used trajectory and pseudo-time analysis to show that cells uniquely basal are plastic in lineage identity and can evolve to form tumor populations composed of other lineage marker-defined signatures. Finally, pathway and drug enrichment analysis of tumor cell clusters were used to identify therapeutics that may preferentially target the identified tumor cell populations. These data collectively define a molecular template that may uniquely profile molecular plasticity occurring during progression and response to therapy with important implications for human disease. SignificanceTumor heterogeneity is a mechanism for treatment resistance. Our study defines unique tumor subpopulations having differential therapeutic sensitivities and potential for lineage plasticity. Our modeling may impact the treatment of BLCA patients.

cancer biology↗

bioRxiv2024

Spatial multi-omics defines a shared infiltrative signature in glioblastoma at the resection margin

Glioblastoma (GBM) remains an untreatable disease. Understanding GBMs infiltrative biology at the resection margin is limited, despite causing disease recurrence and progression. To address this, we generated a high-throughput single-nucleus (sn)RNA-seq and snATAC-seq multi-omic dataset from six tumors with distinct genomic drivers and combined it with spatial transcriptomics to characterize the unique molecular phenotype of GBM near the margin. By contrasting GBM-specific biology in matching "Core" vs. "Margin" dissections, we define unique, shared "GBM infiltration" and chromatin accessibility signatures near the margin. We prioritize EGFR as a top differentially expressed and accessible "Margin" marker across GBM subtypes, show its dynamic expression along a core-to-margin infiltration trajectory, and validate its role in migration through CRISPR/Cas9 deletion in two patient-derived models. ChIP-seq studies furthermore corroborate preferential TEAD1 binding at EGFRs accessible regulatory elements. This validated multi-omic dataset enables further studies into tumor and microenvironment biology in the context of residual GBM disease.

cancer biology↗

bioRxiv2024

Single cell view of tumor microenvironment gradients in pleural mesothelioma

Immunotherapies have shown great promise in pleural mesothelioma (PM), yet most patients still do not achieve significant clinical response, highlighting the importance of improving understanding of the tumor microenvironment (TME). Here, we utilized high-throughput, single-cell RNA-sequencing to de novo identify 54 expression programs and construct a comprehensive cellular catalogue of the PM TME. We found four cancer-intrinsic programs associated with poor disease outcome and a novel fetal-like, endothelial cell population that likely responds to VEGF signaling and promotes angiogenesis. Throughout cellular compartments, we observe substantial difference in the TME associated with a cancer-intrinsic sarcomatoid signature, including enrichment in fetal-like endothelial cells, CXCL9+ macrophages, cytotoxic, exhausted, and regulatory T cells, which we validated using imaging and bulk deconvolution analyses on two independent cohorts. Finally, we show, both computationally and experimentally, that NKG2A-HLA-E interaction between NK and tumor cells represents an important new therapeutic axis in PM, especially for epithelioid cases. Statement of SignificanceThis manuscript presents the first single-cell RNA-sequencing atlas of pleural mesothelioma (PM) tumor microenvironment. Findings of translational relevance, validated experimentally and using independent bulk cohorts, include identification of gene programs predictive of survival, a fetal-like endothelial cell population, and NKG2A blockade as a promising new immunotherapeutic intervention in PM.

cancer biology↗

bioRxiv2024

Intervertebral disc cells from human back pain subjects exhibit TNFR1-mediated senescence and lack TNFR2-modulated repair capacity

Poor intervertebral disc (IVD) healing causes IVD degeneration (IVDD) and progression to herniation and back pain. This study identified distinct roles of TNF-receptors (TNFRs) in contributing to poor healing in painful IVDD. We first isolated IVDD tissue of back pain subjects and determined the complex pro-inflammatory mixture contained many chemokines for recruiting inflammatory cells. Single-cell RNA-sequencing of human IVDD tissues revealed these pro- inflammatory cytokines were dominantly expressed by a small macrophage-population. Human annulus fibrosus (hAF) cells treated with IVDD-conditioned media (CM) underwent senescence with greatly reduced metabolic rates and limited inflammatory responses. TNFR1 inhibition partially restored hAF cell metabolism sufficiently to enable a robust chemokine and cytokine response to CM. We showed that the pro-reparative TNFR2 was very limited on hIVD cell membranes so that TNFR2 inhibition with blocking antibodies or activation using Atsttrin had no effect on hAF cells with CM challenge. However, TNFR2 was expressed in high levels on macrophages identified in scRNA-seq analyses, suggesting their role in repair responses. Results therefore point to therapeutic strategies for painful IVDD involving immunomodulation of TNFR1 signaling in IVD cells to enhance metabolism and enable a more robust inflammatory response including recruitment or delivery of TNFR2 expressing immune cells to enhance IVD repair. SUMMARY STATEMENTTNFR1 signaling drives cells towards senesce and muted inflammatory response in painful intervertebral disc degeneration, while limited TNFR2 signaling may limit disc cell repair responses.

bioengineering↗

bioRxiv2023

Sexually dimorphic role of diet and stress on behavior, energy metabolism, and the ventromedial hypothalamus

Scientific evidence underscores the influence of biological sex on the interplay between stress and metabolic dysfunctions. However, there is limited understanding of how diet and stress jointly contribute to metabolic dysregulation in both males and females. To address this gap, our study aimed to investigate the combined effects of a high-fat diet (HFD) and repeated footshock stress on fear-related behaviors and metabolic outcomes in male and female mice. Using a robust rodent model that recapitulates key aspects of post-traumatic stress disorder (PTSD), we subjected mice to footshock stressor followed by weekly reminder footshock stressor or no stressor for 14 weeks while on either an HFD or chow diet. Our findings revealed that HFD impaired fear memory extinction in male mice that received initial stressor but not in female mice. Blood glucose levels were influenced by both diet and sex, with HFD-fed female mice displaying elevated levels that returned to baseline in the absence of stress, a pattern not observed in male mice. Male mice on HFD exhibited higher energy expenditure, while HFD-fed female mice showed a decreased respiratory exchange ratio (RER). Sex-specific alterations in pro-inflammatory markers and abundance of hematopoietic stem cells were observed in chronically stressed mice on an HFD in different peripheral tissues, indicating the manifestation of distinct comorbid disorders. Single-nuclei RNA sequencing of the ventromedial hypothalamus from stressed mice on an HFD provided insights into sex-specific glial cell activation and cell-type-specific transcriptomic changes. In conclusion, our study offers a comprehensive understanding of the intricate interactions between stress, diet, sex, and various physiological and behavioral outcomes, shedding light on a potential brain region coordinating these interactions.

neuroscience↗

bioRxiv2023

Single Cell RNA-Seq Analysis of Regenerative Drug-Treated Human Pancreatic Islets Identifies A Cycling Alpha Cell Population As Key Beta Cell Progenitors

Diabetes ultimately results from an inadequate number of functional, insulin-producing human beta cells. Although current attempts to replenish the remaining beta cell pool in people with diabetes are encouraging, scalability and cost limit access for the millions of people with diabetes. The small molecule DYRK1A inhibitor class of beta cell regenerative drugs, either alone or in combination with GLP1 receptor agonists or TGF{beta} superfamily inhibitors, are capable of inducing beta cell replication in vitro and increasing beta cell mass in vivo. Despite these advances, the precise mechanisms of action of DYRK1A inhibitors remain incompletely understood. To address the mechanisms more deeply, we performed single cell RNA sequencing on human pancreatic islets treated with a DYRK1A inhibitor, either alone, or in combination with a GLP1 receptor agonist or a TGF{beta} superfamily inhibitor. We identify a cluster of Cycling Alpha Cells as the cells most responsive to DYRK1A inhibition. Velocity and pseudotime lineage trajectory analyses suggest that Cycling Alpha Cells serve as the primary target cell type for of DYRK1A inhibitors, and may serve as precursor cells that transdifferentiate into functional human beta cells in response to the DYRK1A inhibition. In addition to providing a novel mechanism of action for DYRK1A inhibitors, our findings suggest that efforts to target regenerative drugs to human beta cells may be mis-directed: the proper target may be Cycling Alpha Cells.

cell biology↗

bioRxiv2022

Bat pluripotent stem cells reveal unique entanglement between host and viruses

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat (Rhinolophus ferrumequinum) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats unique traits.

developmental biology↗

bioRxiv2022

Single-cell Transcriptomic Atlas of the Human Substantia Nigra in Parkinson's Disease

Parkinsons disease (PD) is a common and complex neurodegenerative disorder. Loss of neuromelanin-containing dopaminergic (DA) neurons in the substantia nigra (SN) is a hallmark of PD neuropathology; the etiology of PD remains unclear. Single-cell (-nucleus) RNA sequencing (sc or snRNAseq) has significantly advanced our understanding of neurodegenerative diseases including Alzheimers, but limited progress has been made in PD. Here we generated by far the largest snRNAseq data of high-quality 315,867 nuclei from the human SN including 9 healthy controls and 23 idiopathic PD cases across different Braak stages. Clustering analysis identified major brain cell types including DA neurons, excitatory neurons, inhibitory neurons, glial cells, endothelial, pericytes, fibroblast and T-cells in the human SN. By combining immunostaining and validating against the datasets from independent cohorts, we identified three molecularly distinct subtypes of DA-related neurons, including a RIT2-enriched population, in human aged SN. All DA neuron subtypes degenerated in PD, whereas the composition of non-neuronal cell clusters including major glial types showed little change. Our study delineated cell-type-specific PD-linked gene expression in the SN and their alterations in PD. Examination of cell-type-based transcriptomic changes suggests the complexity and diversity of molecular mechanisms of PD. Analysis of the remaining DA neurons of the three subtypes from PD demonstrated alterations of common gene sets associated with neuroprotection. Our findings highlight the heterogeneity of DA neurons in the human SN and suggest molecular basis for vulnerability and resilience of human DA neurons in PD. Our cohort thus provides a valuable resource for dissecting detailed mechanisms of DA neuron degeneration and identifying new neuroprotective strategies for PD.

neuroscience↗

bioRxiv2022

Elevated HLA-E and NKG2A as a consequence of chronic immune activation defines resistance to M. bovis BCG immunotherapy in Non-Muscle-Invasive Bladder Cancer

Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the first-line treatment for non-muscle invasive bladder cancer (NMIBC), promotes the production of inflammatory cytokines, particularly interferon (IFN)-{gamma}. Prolonged inflammation and IFN-{gamma} exposure are known to cause an adaptive immune response, enabling immune escape and proliferation by tumor cells. We investigated HLA-E and NKG2A, a novel T and NK cell checkpoint pathway, as a driver of adaptive resistance in BCG unresponsive NMIBC. We observed ubiquitous inflammation in all patients after BCG immunotherapy, regardless of recurrence status. IFN-{gamma} was shown to drive tumor expression of HLA-E and PD-L1. Further, NKG2A-expressing NK and CD8 T cells were enriched in BCG unresponsive tumors and with enhanced capacity for cytolytic functions. Strikingly, in situ spatial analyses revealed that HLA-EHIGH tumors are activated to recruit NK and T cells via chemokine production, potentially sparing HLA-ELOW tumors that would otherwise be susceptible to lysis. Finally, blood-derived NK cells retained anti-tumor functions at the time of tumor recurrence. These data support combined NKG2A and PD-L1 blockade for BCG unresponsive disease.

immunology↗

bioRxiv2021

Drug specific transcriptomic signatures in healthy human subject iPSC derived cardiomyocytes

Drug-induced gene expression profiles can identify potential mechanisms of toxicity. We focused on obtaining signatures for cardiotoxicity of FDA-approved tyrosine kinase inhibitors (TKIs) in human induced pluripotent stem cell-derived cardiomyocytes. Using bulk transcriptomics profiles, we applied singular value decomposition to identify drug-selective patterns in cell lines obtained from multiple healthy human subjects. Cellular pathways affected by highly cardiotoxic TKIs include energy metabolism, contractile, and extracellular matrix dynamics. Projecting these pathways to single cell expression profiles indicates that TKI responses can be evoked in both cardiomyocytes and fibroblasts. Whole genome sequences of the cell lines, using outlier responses enabled us to correctly reidentify a genomic variant associated with anthracycline cardiotoxicity and predict genomic variants potentially associated with TKI cardiotoxicity. We conclude that mRNA expression profiles when integrated with publicly available genomic, pathway, and single cell transcriptomic datasets, provide multiscale predictive understanding of cardiotoxicity for drug development and patient stratification. One sentence summaryGenes, pathways, and cell types of the human heart associated with antineoplastic drug cardiotoxicity.

systems biology↗

bioRxiv2021

Understanding Mechanisms of Chamber-Specific Differentiation Through Combination of Lineage Tracing and Single Cell Transcriptomics

The specification and differentiation of atrial and ventricular myocardial cell types during development is incompletely understood. We have previously shown that Foxa2 expression during gastrulation identifies a population of ventricular fated progenitors, allowing for labeling of these cells prior to the morphogenetic events that lead to chamber formation and acquisition of bona fide atrial or ventricular identity. In this study, we performed single cell RNA sequencing of Foxa2Cre;mTmG embryos at the cardiac crescent (E8.25), primitive heart tube (E8.75) and heart tube (E9.25) stage in order to understand the transcriptional mechanisms underlying formation of atrial and ventricular cell types at the earliest stages of cardiac development. We find that progression towards differentiated myocardial cell types occurs primarily based on heart field progenitor identity, and that different progenitor populations contribute to ventricular or atrial identity through separate differentiation mechanisms. We identified a number of candidate markers that define such differentiation processes, as well as differential regulation of metabolic processes that distinguish atrial and ventricular fated cells at the earliest stages of development. We further show that exogenous injection with retinoic acid during formation of the cardiac primordia causes defects in ventricular chamber size and is associated with dysregulation in FGF signaling in anterior second heart field cells and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit in myocardial committed progenitors that result in formation of hypomorphic ventricles with decreased expression of important metabolic processes and sarcomere assembly. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac progenitor cell specification and differentiation, and the precise effects of manipulating cardiac progenitor field patterning via exogenous retinoic acid signaling.

developmental biology↗

bioRxiv2021

PPARdelta signaling activation improves metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease. A major caveat however is that they remain functionally and structurally immature in culture, limiting their potential for disease modeling and regenerative approaches. Here, we address the question of how different metabolic pathways can be modulated in order to induce efficient hPSC-CM maturation. We show that PPAR signaling acts in an isoform-specific manner to balance glycolysis and fatty acid oxidation (FAO). PPARD activation or inhibition results in efficient respective up- or down-regulation of the gene regulatory networks underlying FAO in hPSC-CMs. PPARD induction further increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation and augments FAO flux. Lastly PPARD activation results in enhanced myofibril organization and improved contractility. Transient lactate exposure, commonly used in hPSC-CM purification protocols, induces an independent program of cardiac maturation, but when combined with PPARD activation equally results in a metabolic switch to FAO. In summary, we identify multiple axes of metabolic modifications of hPSC-CMs and a role for PPARD signaling in inducing the metabolic switch to FAO in hPSC-CMs. Our findings provide new and easily implemented opportunities to generate mature hPSC-CMs for disease modeling and regenerative therapy.

cell biology↗