Biology subjects

Gelb, B. D.

Publications and source records attributed to Gelb, B. D..

15 recordsLinked to original sources

bioRxiv2026

Feeder-Free Generation of Lymphatic Endothelial Cells from Human Induced Pluripotent Stem Cells

AbstractThe lymphatic system is essential for maintaining fluid homeostasis, lipid transport and supporting immune function. Despite its central role in health and disease, advancements in understanding human lymphatic vasculature has been constrained, in part because primary human LECs are difficult to access and study in disease-relevant contexts. This study describes an efficient and scalable feeder-free method to differentiate human iPSCs into lymphatic endothelial cells (LECs) that are transcriptionally and phenotypically similar to primary fetal LECs. An iPSC-derived LEC system overcomes a drawback of primary cells by enabling precise genetic perturbations, supporting study of lymphatic diseases of interest in a human context. By grounding our approach in in vivo stages of lymphangiogenisis, we describe a staged protocol that recapitulates the key milestones of lymphatic development. We first adapted a published method to differentiate human iPSCs into venous endothelial cells (VECs) and then initiate transdifferentiation of VECs into LECs. Using immunocytochemistry, qPCR, as well as flow cytometry, we demonstrated expression of lymphatic-specific markers in the differentiated population. We further characterized our induced VECs (iVECs) and LECs (iLECs) through bulk RNA sequencing analysis and compared the populations to pseudobulk VEC and LEC transcriptomic datasets generated from human fetal heart endothelia at 12, 13 and 14 weeks of gestation. Through this work, we expanded the repertoire of approaches for accessing LECs, with the goal of accelerating discoveries in lymphatic biology and therapeutics. Abstract summary image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/712968v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a9a406org.highwire.dtl.DTLVardef@4faec6org.highwire.dtl.DTLVardef@15b4e73org.highwire.dtl.DTLVardef@17b9c36_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

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

Generation of Valvular Interstitial Cells from Human Pluripotent Stem Cells

Heart valves are living structures whose sophisticated functions are mediated by a specialized population of mesenchymal cells known as valvular interstitial cells (VICs). Given their central role in valve homeostasis, VICs represent a promising cell population for studying heart valve diseases and developing novel therapies to treat them. Here, we describe a strategy for generating VICs from human pluripotent stem cells (hPSCs) by stage-specific manipulation of developmental signalling pathways. Our results demonstrate that hPSC-derived VICs show a high transcriptional similarity to primary human fetal VICs and can secrete key proteins of the valve extracellular matrix. We further investigate the heterogeneity of hPSC-derived VICs and identify two major subpopulations with distinct molecular and functional properties, mirroring the cellular diversity observed in vivo. Finally, we utilize an in vitro model of Noonan syndrome to demonstrate that hPSC-derived VICs can accurately recapitulate key aspects of valve disease. Collectively, these findings provide a reproducible method for the scaled generation of bona fide hPSC-derived VICs and establish their utility in disease modelling and tissue engineering applications. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- We established a robust platform to generate bona fide valvular interstitial cells (VICs) from human pluripotent stem cells (hPSCs), recapitulating native VIC identity and function. - We delineated signaling pathways that promote the development of two distinct VIC subsets and identified a surface marker to distinguish between them. What are the clinical implications?- A renewable, human-specific source of VICs enables precise mechanistic studies of valve development and disease that are not possible with limited surgical specimens or animal models. - This platform creates opportunities for therapeutic applications, including drug discovery and tissue engineering approaches for valve repair.

cell biology↗

bioRxiv2024

Rigosertib reverses hypertrophic cardiomyopathy in RAF1-associated Noonan syndrome

BackgroundRASopathies constitute a group of rare genetic disorders caused by mutations in genes that reside along the canonical Ras/MAPK signaling pathway, affecting cell growth and differentiation. These syndromes, which include conditions like Noonan syndrome (NS), are characterized by developmental delays, distinctive facial dysmorphia, and a variety of cardiac defects, notably hypertrophic cardiomyopathy (HCM). Despite their prevalence and impact, therapeutic options for RASopathies remain limited. Rigosertib, a novel dual Ras/MAPK and PI3K/AKT pathway inhibitor, is currently in clinical trials for treatment of melanoma and recessive dystrophic epidermolysis bullosa. Here, we identify rigosertib as a candidate therapy for RAF1-associated HCM. Methods and ResultsOur Drosophila screen of clinically relevant drugs and compounds identified rigosertib as broadly effective across a panel of transgenic RASopathy fly transgenic models, indicating that rigosertib may be effective against multiple disease isoforms. Analysis of a Drosophila model targeting a RAF1L613V transgene to the heart found that rigosertib reduced aspects of cardiac hypertrophy. Rigosertib treatment prevented or regressed cellular hypertrophy in human induced pluripotent stem cell-(iPSC-) derived cardiomyocytes homozygous for the NS-associated RAF1S257L allele. We extended these findings to a mammalian model, using Raf1L613V/+ KI mice to explore the therapeutic implications of rigosertib on RAF1-driven HCM. Longitudinal six-week treatment with rigosertib in these mice resulted in significant improvement in left ventricular chamber dimension and posterior wall thickness, total heart mass, size of individual cardiomyocytes (CMs), as well as reversal of cardiac hypertrophy. Rigosertib treatment also led to normalized fetal gene expression and inhibition of ERK and AKT pathway activities in primary CMs isolated from Raf1L613V/+ mice. Cardiac function, as assessed by echocardiography, showed significant improvement in ejection fraction and fractional shortening, with molecular studies confirming downregulation of hypertrophic markers and signaling pathways. Together with the Drosophila data, these mammalian results support the potential and use for rigosertib to reverse pathological hypertrophy in NS through targeted pathway inhibition in patients. Moreover, in addition to its effects in the heart, rigosertib treatment in mice also significantly improved other NS-associated syndromic features, including increasing bone growth and correcting craniofacial abnormalities. ConclusionsTaken together, our findings suggest rigosertib effectively normalizes and reverses RASopathy-associated HCM as well as other NS-associated syndromic features, supporting its potential for development as a promising treatment for RAF1-associated HCM and, potentially, other RASopathies-dependent pathologies. This study not only highlights the therapeutic potential of rigosertib but also demonstrates the utility of an integrated approach using Drosophila, iPSC and mammalian models to elucidate drug effects across complex biological systems.

molecular biology↗

bioRxiv2024

Automated Identification of Germline de novo Mutations in Family Trios: A Consensus-Based Informatic Approach

Accurate identification of germline de novo variants (DNVs) remains a challenging problem despite rapid advances in sequencing technologies as well as methods for the analysis of the data they generate, with putative solutions often involving ad hoc filters and visual inspection of identified variants. Here, we present a purely informatic method for the identification of DNVs by analyzing short-read genome sequencing data from proband-parent trios. Our method evaluates variant calls generated by three genome sequence analysis pipelines utilizing different algorithms--GATK HaplotypeCaller, DeepTrio and Velsera GRAF--exploring the assumption that a requirement of consensus can serve as an effective filter for high- quality DNVs. We assessed the efficacy of our method by testing DNVs identified using a previously established, highly accurate classification procedure that partially relied on manual inspection and used Sanger sequencing to validate a DNV subset comprising less confident calls. The results show that our method is highly precise and that applying a force-calling procedure to putative variants further removes false-positive calls, increasing precision of the workflow to 99.6%. Our method also identified novel DNVs, 87% of which were validated, indicating it offers a higher recall rate without compromising accuracy. We have implemented this method as an automated bioinformatics workflow suitable for large- scale analyses without need for manual intervention.

bioinformatics↗

bioRxiv2023

Feeder-Free Generation of Endocardial and Cardiac Valve Cells from Human Pluripotent Stem Cells

Valvular heart disease presents a significant health burden, yet advancements in valve biology and novel therapeutics have been hindered by the lack of accessibility to human valve cells. In this study, we have developed a scalable and feeder-free method to differentiate human induced pluripotent stem cells (iPSCs) into endocardial cells. Importantly, we show that these endocardial cells are transcriptionally and phenotypically distinct from vascular endothelial cells and can be directed to undergo endothelial-to-mesenchymal transition (EndMT) to generate cardiac valve cell populations. Following this, we identified two distinct populations--one population undergoes EndMT to become valvular interstitial cells (VICs), while the other population reinforces their endothelial identity to become valvular endothelial cells (VECs). Lastly, we confirmed the identities of our iPSC-derived cell populations and identified putative markers through transcriptomic analyses. By increasing the accessibility to these cell populations, we aim to accelerate discoveries for cardiac valve biology and disease.

cell biology↗

bioRxiv2023

Structural variation across 138,134 samples in the TOPMed consortium

Ever larger Structural Variant (SV) catalogs highlighting the diversity within and between populations help researchers better understand the links between SVs and disease. The identification of SVs from DNA sequence data is non-trivial and requires a balance between comprehensiveness and precision. Here we present a catalog of 355,667 SVs (59.34% novel) across autosomes and the X chromosome (50bp+) from 138,134 individuals in the diverse TOPMed consortium. We describe our methodologies for SV inference resulting in high variant quality and >90% allele concordance compared to long-read de-novo assemblies of well-characterized control samples. We demonstrate utility through significant associations between SVs and important various cardio-metabolic and hemotologic traits. We have identified 690 SV hotspots and deserts and those that potentially impact the regulation of medically relevant genes. This catalog characterizes SVs across multiple populations and will serve as a valuable tool to understand the impact of SV on disease development and progression.

genomics↗

bioRxiv2022

Alteration of myocardial structure and function in RAF1associated Noonan syndrome: Insights from cardiac disease modeling based on patient-derived iPSCs

Noonan syndrome (NS), the most common among the RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770C>T missense change. We characterize the molecular, structural and functional consequences of aberrant RAF1 -associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes, and myocardial tissue biopsies. The disease phenotype was partly reverted by using both MEK inhibition, and a gene-corrected isogenic RAF1L257S cell line. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease. These insights represent a basis to develop future targeted therapeutic approaches.

cell biology↗

bioRxiv2021

Integration of Protein Interactome Networks with Congenital Heart Disease Variants Reveals Candidate Disease Genes

Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains a challenge despite large-scale genomic sequencing efforts. We hypothesized that genetic determinants for CHDs may lie in protein interactomes of GATA4 and TBX5, two transcription factors that cause CHDs. Defining their interactomes in human cardiac progenitors via affinity purification-mass spectrometry and integrating results with genetic data from the Pediatric Cardiac Genomic Consortium revealed an enrichment of de novo variants among proteins that interact with GATA4 or TBX5. A consolidative score that prioritized interactome members based on variant, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied cardiac developmental genes, resulting in co-activation, and the GLYR1 missense variant associated with CHD disrupted interaction with GATA4. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating the contribution of genetic variants in disease.

genetics↗

bioRxiv2020

Whole Genome De Novo Variant Identification with FreeBayes and Neural Network Approaches

MotivationDe novo variant (DNV) calling typically relies on heuristic filters intrinsic to specific platforms and variant calling algorithms. FreeBayes and neural network approaches have overcome this limitation for variant calling, and we implemented a similar approach for DNV identification. ResultsWe developed a DNV calling framework that uses Genome Analysis Toolkit (GATK), FreeBayes and a neural network trained on Integrative Genomics Viewer pile-up plots (IGV-bot). We identified DNVs in 2,390 WGS trios and benchmarked results against heuristics based on GATK parameters. Results were validated in silico and with Sanger sequencing, with the latter showing true positive rates of 98.4% and 97.3% for SNVs and indels, respectively. Taken together we describe a scalable framework for DNV identification based on both FreeBayes and neural network methods. AvailabilitySource code and documentation are available at https://github.com/ShenLab/igv-classifier and https://github.com/frichter/dnv_pipeline under the MIT license. Contactys2411@cumc.columbia.edu

genomics↗

bioRxiv2019

Early post-zygotic mutations contribute to congenital heart disease

BackgroundThe contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined.\n\nResultsWe developed a computational method, Expectation-Maximization-based detection of Mosaicism (EM-mosaic), to analyze mosaicism in exome sequences of 2530 CHD proband-parent trios. EM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The frequency of mosaic variants above 10% mosaicism was 0.13/person in blood and 0.14/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction.\n\nConclusionsWe estimate that ~1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants expressed at higher allele fraction compared to benign variants. Although blood is a readily-available DNA source, cardiac tissues analyzed contributed ~5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses.

bioinformatics↗

bioRxiv2018

Assessing the Gene-Disease Association of 19 Genes with the RASopathies using the ClinGen Gene Curation Framework

The RASopathies are a complex group of diseases regarding phenotype and genetic etiology. The ClinGen RASopathy Expert Panel assessed published and other publicly available evidence supporting the association of 19 genes with RASopathy conditions. Using the semi-quantitative literature curation method developed by the ClinGen Gene Curation Working Group, evidence for each gene was curated and scored for Noonan syndrome, Costello syndrome, cardiofaciocutaneous (CFC) syndrome, Noonan syndrome with multiple lentigines (NSML), and Noonan-like syndrome with loose anagen hair (NS/LAH).\n\nThe curated evidence supporting each gene-disease relationship was then discussed and approved by the ClinGen RASopathy Expert Panel. Each associations strength was classified as Definitive, Strong, Moderate, Limited, Disputed, or No Evidence. Eleven genes were classified as definitively associated with at least one RASopathy condition. Two genes classified as strong for association with at least one RASopathy condition while one gene was moderate and three were limited. The RAS EP also refuted the association of two genes for a RASopathy condition. Overall, our results provide a greater understanding of the different gene-disease relationships within the RASopathies and can help guide and direct clinicians, patients and researchers who are identifying variants in individuals with a suspected RASopathy\n\nGRANT NUMBERSResearch reported in this publication was supported by the National Human Genome Research Institute (NHGRI) under award number U41HG006834. MZ received support from German Federal Ministry of Education and Research (BMBF): NSEuroNet (FKZ 01GM1602A), GeNeRARe (FKZ 01GM1519A).

genetics↗

bioRxiv2018

Identification of rare de novo epigenetic variations in congenital disorders

Certain human traits such as neurodevelopmental disorders (NDs) and congenital anomalies (CAs) are believed to be primarily genetic in origin. With recent dramatic advances in genomic technologies, genome-wide surveys of cohorts of patients with ND/CAs for point mutations and structural variations have greatly advanced our understanding of their genetic etiologies1,2. However, even after whole genome sequencing (WGS), a substantial fraction of such disorders remain unexplained3. In contrast, the possibility that constitutive epigenetic variations (epivariations) might underlie such traits has not been well explored. We hypothesized that some cases of ND/CA are caused by aberrations of DNA methylation that lead to a dysregulation of normal genome function. By comparing DNA methylation profiles from 489 individuals with ND/CAs against 1,534 population controls, we identified epivariations as a frequent occurrence in the human genome. De novo epivariations were significantly enriched in cases when compared to controls. RNAseq data from population studies showed that epivariations often have an impact on gene expression comparable to loss-of-function mutations. Additionally, we detected and replicated an enrichment of rare sequence mutations overlapping CTCF binding sites close to epivariations. Thus, some epivariations occur secondary to cis-linked mutations in regulatory regions, providing a rationale for interpreting non-coding genetic variation. We propose that epivariations likely represent the causative genomic defect in 5-10% of patients with unexplained ND/CAs. This constitutes a yield comparable to CNV microarrays, and as such has significant diagnostic relevance.

genomics↗

bioRxiv2017

Robust identification of deletions in exome and genome sequence data based on clustering of Mendelian errors

Multiple tools have been developed to identify copy number variants (CNVs) from whole exome (WES) and whole genome sequencing (WGS) data. Current tools such as XHMM for WES and CNVnator for WGS identify CNVs based on changes in read depth. For WGS, other methods to identify CNVs include utilizing discordant read pairs and split reads and genome-wide local assembly with tools such as Lumpy and SvABA, respectively. Here, we introduce a new method to identify deletion CNVs from WES and WGS trio data based on the clustering of Mendelian errors (MEs). Using our Mendelian Error Method (MEM), we identified 127 deletions (inherited and de novo) in 2,601 WES trios from the Pediatric Cardiac Genomics Consortium, with a validation rate of 88% by digital droplet PCR. MEM identified additional de novo deletions compared to XHMM, and also identified sample switches, DNA contamination, a significant enrichment of 15q11.2 deletions compared to controls and eight cases of uniparental disomy. We applied MEM to WGS data from the Genome In A Bottle Ashkenazi trio and identified deletions with 97% specificity. MEM provides a robust, computationally inexpensive method for identifying deletions, and an orthogonal approach for verifying deletions called by other tools.

genetics↗

bioRxiv2017

Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo

Epigenetic mechanisms and transcription factor networks essential for differentiation of cardiac myocytes have been uncovered. However, reshaping of the epigenome of these terminally differentiated cells during fetal development, postnatal maturation and in disease remains unknown. Thus, the aim of this study was to determine the dynamics of the cardiac myocyte epigenome during development and in chronic heart failure. Prenatal development and postnatal maturation are characterized by a cooperation of active CpG methylation and histone marks at cis-regulatory and genic regions to shape the cardiac myocyte transcriptome. In contrast, pathological gene expression in terminal heart failure is accompanied by changes in active histone marks without major alterations in CpG methylation and repressive chromatin marks. Notably, cis-regulatory regions in cardiac myocytes are significantly enriched for cardiovascular disease-associated variants. This study uncovers distinct layers of epigenetic regulation not only during prenatal development and postnatal maturation but also in diseased human cardiac myocytes.

genomics↗