Biology subjects

Bertero, A.

Publications and source records attributed to Bertero, A..

17 recordsLinked to original sources

bioRxiv2026

Genetic control couples TGFβ-independent pluripotent stem cell renewal to deterministic forward programming across germ layers

Reproducible and scalable manufacture of human induced pluripotent stem cell (hiPSC) derivatives requires control over expansion and differentiation. Transcription factor-mediated forward programming can place hiPSC differentiation under genetic control, but expansion still relies on extracellular growth-factor supplementation, increasing process complexity and operator dependence. Here we introduce GERALT (Genetically Enhanced Renewal and Autonomous Lineage Transdifferentiation), a push-pull genetic circuit assembled through targeted safe-harbour integration that couples NANOG-supported, TGF{beta}-independent renewal to doxycycline-inducible lineage programming and simultaneous NANOG silencing. GERALT maintained pluripotency during extended TGF{beta} withdrawal in both adherent and suspension culture, yet remained programmable towards myogenic, neuronal, and hepatic fates. Compared with the established OPTi-OX platform in myogenesis, GERALT enabled differentiation in simplified medium while canalising and accelerating commitment. GERALT also supported direct myogenic programming in suspension and enabled direct casting of hiPSCs into hydrogel followed by in-tissue programming, yielding electrically responsive engineered muscle tissues. These results extend genetic control upstream into stem cell renewal, facilitating deployment of new approach methodologies and other biomanufacturing applications.

synthetic biology↗

bioRxiv2026

Combinatorial and Inducible CRISPRa/i Enables Canalized hiPSC Forward Programming and Iterative Refinement via Single-Cell Genomics

Synthetic gene-regulation logic is established in immortalized cell lines but remains largely aspirational in human induced pluripotent stem cells (hiPSCs) and derivatives. This gap constrains both mechanistic discovery and translational engineering in physiologically relevant models. We developed CIRI (Combinatorial Inducible CRISPR in IPSCs), an isogenic, safe-harbor-engineered platform in which tetracycline-responsive single guide RNAs (sgR-NAs) carry modular RNA aptamers that recruit RNA-binding proteins and effector domains. This design enables multimodal regulation from a single catalytically inactive Cas9 (dCas9), exemplified by orthogonal CRISPR activation and interference (CRISPRa/i). After optimizing sgRNA-aptamer architectures, we achieved robust CRISPRa and CRISPRi in hiPSCs and hiPSC-derived cardiac organoids. CIRI rapidly channels hiPSC forward programming into skeletal myocytes by activating MYOD1 while repressing NANOG, POU5F1/OCT4, and SOX2. Combinatorial pooled dual-guide single-cell RNA sequencing screens identify ID3 as a road-block and KDM6B and SMARCD3 as synergistic enhancers of myogenic maturation. Together, CIRI establishes a programmable synthetic biology framework in human stem cell models. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/729073v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@18ce445org.highwire.dtl.DTLVardef@de8d94org.highwire.dtl.DTLVardef@1212763org.highwire.dtl.DTLVardef@1a0d83d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Opposing CTCF and GATA4 activities set the pace of chromatin topology remodeling during cardiomyogenesis

Reorganization of the three-dimensional chromatin structure is a critical feature of human embryonic development. Yet, the mechanisms regulating integrative remodelling of local structures (e.g., loops) and global architecture (e.g., A/B compartmentalization) remain un-clear. Here, we investigate this aspect in the context of cardiomyogenesis, characterized by pronounced B-to-A remodelling of several cardiac-specific genes such as TTN. We focus on the roles of the pioneer transcription factor GATA4 and the architectural protein CTCF. Using an inducible knockdown system during human induced pluripotent stem cell differ-entiation, we show that GATA4 is essential for timely topological activation of key cardiac genes, while partial depletion of CTCF, anticipating physiological downregulation during de-velopment, enhances this process. Deletion of a single CTCF binding site on TTN leads to modest gene decompaction and transcriptional activation. Bulk and single-cell RNA se-quencing of chamber-specific cardiac organoids reveals that loss of GATA4 delays differ-entiation and sustains proliferation of early cardiomyocytes, whereas premature CTCF de-pletion accelerates yet alters cardiomyocyte maturation. These findings suggest that CTCF and GATA4 have antagonistic roles on chromatin dynamics during cardiomyogenesis, form-ing a rheostat that maintains accurate developmental tempo. Disruption of this mecha-nism may contribute to congenital heart defects caused by mutations in these factors.

molecular biology↗

bioRxiv2024

CALIPERS: Cell cycle-aware live imaging for phenotyping experiments and regeneration studies

Cell cycle progression, migration, and proliferation shape development and regeneration, but simultaneous live-cell imaging remains challenging as conventional fluorescent cell cycle indicators (FUCCI) monopolize the green and red channels used by most structural and functional biosensors. To overcome this, we integrated a spectrally re-engineered FUCCI variant, open-source analysis software, and four-color human stem cell reporter lines into CALIPERS: a method for Cell-cycle-Aware Live-cell Imaging in Phenotyping and Regeneration Studies.

bioengineering↗

bioRxiv2024

Brain-body circuit by which midbrain dopamine neuronal activity modulates splenic immunity

Midbrain dopamine neurons are well-known to shape central nervous system function, yet there is growing evidence for their influence on the peripheral immune system. Here we demonstrate that midbrain dopamine neurons form a circuit to the spleen via a multi-synaptic pathway from the dorsal vagal complex (DVC) through the celiac ganglion. Midbrain dopamine neurons modulate the activity of D1- and D2-like dopamine receptor-expressing DVC neurons. In vivo activation of midbrain dopamine neurons induced dopamine release in the DVC and increased immediate early gene expression in both the DVC and celiac ganglion. Activation of this midbrain-to-spleen circuit reduced spleen weight and decreased naive CD4+ T-cell populations without affecting total T-cell numbers. These findings unveil a functional midbrain- DVC-celiac ganglion-spleen pathway, through which midbrain dopamine neurons modulate splenic immunity. These novel insights into the neural regulation of the immune system have important implications for diseases involving altered dopamine neurotransmission and highlight potential targets for immunotherapeutic interventions.

neuroscience↗

bioRxiv2024

Nascent transcript O-MAP reveals the molecular architecture of a single-locus subnuclear compartment built by RBM20 and the TTN RNA

Eukaryotic nuclei adopt a highly compartmentalized architecture that influences nearly all genomic processes. Understanding how this architecture impacts gene expression has been hindered by a lack of tools for elucidating the molecular interactions at individual genomic loci. Here, we adapt oligonucleotide-mediated proximity-interactome mapping (O-MAP) to biochemically characterize discrete, micron-scale nuclear neighborhoods. By targeting O-MAP to introns within the TTN pre-mRNA, we systematically map the chromatin loci, RNAs, and proteins within a muscle-specific RNA factory organized around the TTN locus. This reveals an unanticipated compartmental architecture that organizes cis- and trans-interacting chromosomal domains, including a hub of transcriptionally silenced chromatin. The factory also recruits dozens of unique RNA-binding and chromatin-scaffolding factors, including QKI and SAFB, along with their target transcripts. Loss of the cardiac-specific splicing factor RBM20--a master regulator of TTN splicing that is mutated in dilated cardiomyopathy--remodels nearly every facet of this architecture. This establishes O-MAP as a pioneering method for probing single-locus, microcompartment-level interactions that are opaque to conventional tools. Our findings suggest new mechanisms by which coding genes can "moonlight" in nuclear-architectural roles.

genomics↗

bioRxiv2024

Prediction and functional interpretation of inter-chromosomal genome architecture from DNA sequence with TwinC

Three-dimensional nuclear DNA architecture comprises well-studied intra-chromosomal (cis) folding and less characterized inter-chromosomal (trans) interfaces. Current predictive models of 3D genome folding can effectively infer pairwise cis-chromatin interactions from the primary DNA sequence but generally ignore trans contacts. There is an unmet need for robust models of trans-genome organization that provide insights into their underlying principles and functional relevance. We present TwinC, an interpretable convolutional neural network model that reliably predicts trans contacts measurable through proximity ligation-dependent (in situ and intact Hi-C) and independent (DNA SPRITE) genome-wide chromatin conformation assays.. TwinC uses a paired sequence design from replicate Hi-C experiments to learn single base pair relevance in trans interactions across two stretches of DNA. The method achieves high predictive accuracy (AUROC=0.80) on a cross-chromosomal test set from in situ and intact Hi-C experiments in heart tissue. Furthermore, we train TwinC using in situ Hi-C data from the widely used GM12878 cell line and validate its performance with orthogonal DNA SPRITE assay in the same cell type. Mechanistically, the neural network learns the importance of compartments, chromatin accessibility, clustered transcription factor binding and G-quadruplexes in forming trans contacts. In summary, TwinC models and interprets trans genome architecture, shedding light on this poorly understood aspect of gene regulation.

bioinformatics↗

bioRxiv2023

Systematic identification of inter-chromosomal interaction networks supports the existence of RNA factories

Most studies of genome organization have focused on intra-chromosomal (cis) contacts because they harbor key features such as DNA loops and topologically associating domains. Inter-chromosomal (trans) contacts have received much less attention, and tools for interrogating potential biologically relevant trans structures are lacking. Here, we develop a computational framework to identify sets of loci that jointly interact in trans from Hi-C data. This method, trans-C, initiates probabilistic random walks with restarts from a set of seed loci to traverse an input Hi-C contact network, thereby identifying sets of trans-contacting loci. We validate trans-C in three increasingly complex models of established trans contacts: the Plasmodium falciparum var genes, the mouse olfactory receptor "Greek islands", and the human RBM20 cardiac splicing factory. We then apply trans-C to systematically test the hypothesis that genes co-regulated by the same trans-acting element (i.e., a transcription or splicing factor) co-localize in three dimensions to form "RNA factories" that maximize the efficiency and accuracy of RNA biogenesis. We find that many loci with multiple binding sites of the same transcription factor interact with one another in trans, especially those bound by transcription factors with intrinsically disordered domains. Similarly, clustered binding of a subset of RNA binding proteins correlates with trans interaction of the encoding loci. These findings support the existence of trans interacting chromatin domains (TIDs) driven by RNA biogenesis. Trans-C provides an efficient computational framework for studying these and other types of trans interactions, empowering studies of a poorly understood aspect of genome architecture.

genomics↗

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↗

bioRxiv2021

Early anteroposterior regionalisation of human neural crest is shaped by a pro-mesodermal factor

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cells, the presumed precursors of the childhood tumour neuroblastoma, are derived from neuromesodermal-potent progenitors of the postcranial body (NMPs). Here we employ human embryonic stem cell differentiation to define how NMP-derived NC cells acquire a posterior axial identity. We show that TBXT, a pro-mesodermal transcription factor, mediates early posterior NC regionalisation together with WNT signalling effectors. This occurs by TBXT-driven chromatin remodelling via its binding in key enhancers within HOX gene clusters and other posterior regulator-associated loci. In contrast, posteriorisation of NMP-derived spinal cord cells is TBXT/WNT-independent and takes place under the influence of FGF signalling. Our work reveals a previously unknown role of TBXT in influencing posterior NC fate and points to the existence of temporally discrete, cell type-dependent modes of posterior axial identity control.

developmental biology↗

bioRxiv2021

Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein aggregates within processing bodies

RNA binding motif protein 20 (RBM20) is a key regulator of alternative splicing in the heart, and its mutation leads to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous DCM-associated missense mutations in RBM20 (R636S) as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulated contractile dysfunction of RBM20-associated DCM and revealed greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP revealed a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing revealed that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO, impacting distinct cardiac signaling pathways. Splicing defects specific to KO or R636S mutations were supported by data from R636S gene-edited pig hearts and eCLIP. Super-resolution microscopy verified that mutant RBM20 maintains limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a novel pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways that are likely to mediate differential contractile phenotypes and stress-associated heart pathology.

cell biology↗

bioRxiv2021

Pharmacologic Therapy for Engraftment Arrhythmia Induced by Transplantation of Human Cardiomyocytes

BackgroundEngraftment arrhythmias (EAs) are observed in large animal studies of intramyocardial transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) for myocardial infarction. Although transient, the risk posed by EA presents a barrier to clinical translation. ObjectivesWe hypothesized that clinically approved antiarrhythmic drugs can prevent EA-related mortality as well as suppress tachycardia and arrhythmia burden. MethodshPSC-CM were transplanted into the infarcted porcine heart by surgical or percutaneous delivery to induce EA. Following a screen of antiarrhythmic agents, a prospective study was conducted to determine the effectiveness of amiodarone plus ivabradine in preventing cardiac death and suppressing EA. ResultsEA was observed in all subjects, and amiodarone-ivabradine treatment was well-tolerated. None of the treated subjects experienced the primary endpoint of cardiac death, unstable EA or heart failure compared to 5/8 (62.5%) in the control cohort (hazard ratio 0.00; 95% confidence interval, 0-0.297; p = 0.002). Overall survival including two deaths in the treated cohort from immunosuppression-related infection showed borderline improvement with treatment (hazard ratio 0.21; 95% confidence interval, 0.03-1.01; p = 0.05). Without treatment, peak heart rate averaged 305 {+/-} 29 beats per min (bpm), whereas in treated subjects peak daily heart rate was significantly restricted to 185{+/-}9 bpm (p = 0.006). Similarly, treatment reduced peak daily EA burden from 96.8 {+/-} 2.9% to 76.5 {+/-} 7.9% (p = 0.003). Antiarrhythmic treatment was safely discontinued after approximately one-month of treatment without recrudescence of arrhythmia. ConclusionsThe risk of engraftment arrhythmia following hPSC-CM transplantation can be reduced significantly by combined amiodarone and ivabradine drug therapy. Condensed AbstractEngraftment arrhythmia (EA) is a transient but serious complication of cardiac remuscularization therapy. Using a porcine model of cardiac remuscularization and EA, ivabradine and amiodarone were independently effective in suppressing tachycardia and arrhythmia, respectively. Baseline amiodarone combined with adjunctive ivabradine successfully prevented cardiac death, unstable EA and heart failure (hazard ratio 0.00; 95% confidence interval, 0-0.297; p = 0.002) and significantly suppressed peak daily heart rate and arrhythmia burden (p=0.006 and 0.003, respectively). Antiarrhythmic treatment was successfully discontinued after one-month without recrudescence of arrhythmia. We conclude that EA can be suppressed by combined amiodarone and ivabradine drug therapy.

pharmacology and toxicology↗

bioRxiv2020

A cross-species link between mTOR-related synaptic pathology and functional hyperconnectivityin autism

Postmortem studies have revealed increased density of excitatory synapses in the brains of individuals with autism, with a putative link to aberrant mTOR-dependent synaptic pruning. Autism is also characterized by atypical macroscale functional connectivity as measured with resting-state fMRI (rsfMRI). These observations raise the question of whether excess of synapses cause aberrant functional connectivity in autism. Using rsfMRI, electrophysiology and in silico modelling in Tsc2 haploinsufficient mice, we show that mTOR-dependent increased spine density is associated with autism-like stereotypies and cortico-striatal hyperconnectivity. These deficits are completely rescued by pharmacological inhibition of mTOR. Notably, we further demonstrate that children with idiopathic autism exhibit analogous cortical-striatal hyperconnectivity, and document that this connectivity fingerprint is enriched for autism-dysregulated genes interacting with mTOR or TSC2. Finally, we show that the identified transcriptomic signature is predominantly expressed in a subset of children with autism, thereby defining a segregable autism subtype. Our findings causally link mTOR-related synaptic pathology to large-scale network aberrations, revealing a unifying multi-scale framework that mechanistically reconciles developmental synaptopathy and functional hyperconnectivity in autism. SignificanceAberrant brain functional connectivity is a hallmark of autism, but the neural basis of this phenomenon remains unclear. We show that a mouse line recapitulating mTOR-dependent synaptic pruning deficits observed in postmortem autistic brains exhibits widespread functional hyperconnectivity. Importantly, pharmacological normalization of mTOR signalling completely rescues synaptic, behavioral and functional connectivity deficits. We also show that a similar connectivity fingerprint can be isolated in human fMRI scans of people with autism, where it is linked to over-expression of mTOR-related genes. Our results reveal a unifying multi-scale translational framework that mechanistically links aberrations in synaptic pruning with functional hyperconnectivity in autism.

neuroscience↗

bioRxiv2020

SARS-CoV-2 infects human pluripotent stem cell-derived cardiomyocytes, impairing electrical and mechanical function

Global health has been threatened by the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2)1. Although considered primarily a respiratory infection, many COVID-19 patients also suffer severe cardiovascular disease2-4. Improving patient care critically relies on understanding if cardiovascular pathology is caused directly by viral infection of cardiac cells or indirectly via systemic inflammation and/or coagulation abnormalities3,5-9. Here we examine the cardiac tropism of SARS-CoV-2 using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and three-dimensional engineered heart tissues (3D-EHTs). We observe that hPSC-CMs express the viral receptor ACE2 and other viral processing factors, and that SARS-CoV-2 readily infects and replicates within hPSC-CMs, resulting in rapid cell death. Moreover, infected hPSC-CMs show a progressive impairment in both electrophysiological and contractile properties. Thus, COVID-19-related cardiac symptoms likely result from a direct cardiotoxic effect of SARS-CoV-2. Long-term cardiac complications might be possible sequelae in patients who recover from this illness.

pathology↗

bioRxiv2019

Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

Pathogenic mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses (Hi-C) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared to gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape inactivation during cardiogenesis. These regions exhibit upregulation of multiple non-cardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. On the other hand, A/B compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. We conclude that global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency. SummaryBertero et al. observe that lamin A/C haploinsufficiency in human cardiomyocytes markedly alters electrophysiology, contractility, gene expression, and chromosomal topology. Contrary to expectations, however, changes in chromatin compartments involve just few regions, and most dysregulated genes lie outside these hotspots. Condensed titleGenomic effects of lamin A/C haploinsufficiency

genomics↗

bioRxiv2018

Deletion of autism risk gene Shank3 disrupts prefrontal connectivity

Mutations in the synaptic scaffolding protein Shank3 are a major cause of autism, and are associated with prominent intellectual and language deficits. However, the neural mechanisms whereby SHANK3 deficiency affects higher order socio-communicative functions remain unclear. Using high-resolution functional and structural MRI in mice, here we show that loss of Shank3 (Shank3B-/-) results in disrupted local and long-range prefrontal functional connectivity, as well as fronto-striatal decoupling. We document that prefrontal hypo-connectivity is associated with reduced short-range cortical projections density, and reduced gray matter volume. Finally, we show that prefrontal disconnectivity is predictive of social communication deficits, as assessed with ultrasound vocalization recordings. Collectively, our results reveal a critical role of SHANK3 in the development of prefrontal anatomy and function, and suggest that SHANK3 deficiency may predispose to intellectual disability and socio-communicative impairments via dysregulation of higher-order cortical connectivity.

neuroscience↗

bioRxiv2017

Dynamic reorganization of nuclear architecture during human cardiogenesis

While chromosomal architecture varies among cell types, little is known about how this organization is established or its role in development. We integrated Hi-C, RNA-seq and ATAC-seq during cardiac differentiation from human pluripotent stem cells to generate a comprehensive profile of chromosomal architecture. We identified active and repressive domains that are dynamic during cardiogenesis and recapitulate in vivo cardiomyocytes. During differentiation, heterochromatic regions condense in cis. In contrast, many cardiac-specific genes, such as TTN (titin), decompact and transition to an active compartment coincident with upregulation. Moreover, we identify a network of genes, including TTN, that share the heart-specific splicing factor, RBM20, and become associated in trans during differentiation, suggesting the existence of a 3D nuclear splicing factory. Our results demonstrate both the dynamic nature in nuclear architecture and provide insights into how developmental genes are coordinately regulated.\n\nOne Sentence SummaryThe three-dimensional structure of the human genome is dynamically regulated both globally and locally during cardiogenesis.

genomics↗