Biology subjects

Rossello, F. J.

Publications and source records attributed to Rossello, F. J..

10 recordsLinked to original sources

bioRxiv2025

Efficient installation of heterozygous mutations in human pluripotent stem cells using prime editing

The utility of human pluripotent stem cells (hPSCs) is greatly enhanced by the ability to introduce precise, site-specific genetic modifications with minimal off-target effects. Although Cas9 endonuclease is an exceptionally efficient gene-editing tool, its propensity for generating biallelic modifications often limits its capacity for introducing heterozygous variants. Here, we use prime editing (PE) to install heterozygous edits in over ten distinct genetic loci, achieving knock-in efficiencies of up to 40% without the need for subsequent purification or drug selection steps. Moreover, PE enables the precise introduction of heterozygous edits in paralogous genes that are otherwise extremely challenging to achieve using endonuclease-based editing approaches. We also show that PE can be successfully combined with reprogramming to derive heterozygous iPSC clones directly from human fibroblasts and peripheral blood mononuclear cells. Our findings highlight the utility of PE for generating hPSCs with complex edits and represents a powerful platform for modelling disease-associated dominant mutations and gene-dosage effects in an entirely isogenic context.

cell biology↗

bioRxiv2025

Macrophages Mediate Antiviral Immunity and Repair of Type 2 Alveolar Epithelial Cells in a Human Stem Cell Model

The lung alveoli are constantly exposed to inhaled pathogens and inorganic hazards, relying on robust defence mechanisms to maintain homeostasis. Alveolar macrophages and type 2 alveolar epithelial cells (AT2s) collaborate to orchestrate protection. Compromised defence can dysregulate immunity and repair, leading to acute and chronic respiratory diseases. To better understand these processes and drive therapeutic discovery, human model systems that capture key cell interactions are essential. Here, we develop the first induced pluripotent stem cell (iPSC)-derived platform that integrates AT2 cells and macrophages in an air-liquid interface culture. Coculture enhanced AT2-specific gene expression and lipid synthesis, while macrophages actively phagocytosed AT2-derived surfactant. iPSC-derived AT2s supported macrophage survival by producing M-CSF and coculture promoted an alveolar macrophage-like phenotype. Additionally, during respiratory infection macrophages played a crucial role in modulating proinflammatory signalling, enhancing antiviral immunity, and restricting viral replication. Furthermore, we identify a role for iPSC-derived macrophages in epithelial repair, with VEGF signalling to macrophages increasing epithelial permeability. We present an iPSC-derived air-interface platform to study AT2-macrophage interactions in homeostasis, infection, and repair, providing insights into their potential roles in the initiation and progression of respiratory diseases.

cell biology↗

bioRxiv2024

Long-lived chondroprogenitors are generated by fetal-limb Gli1+ cells and are replenished upon mosaic cell-cycle arrest in the cartilage

The growth-plate cartilage of the developing long bones is a well-known system of spatially segregated stem/progenitor, transient amplifying and terminally differentiated cells. However, the regulation of the number and activity of long-lived cartilage progenitors (LLCPs) is poorly understood, despite its relevance for understanding human-height variation, the evolution of limb size and proportions and the aetiology of skeletal growth disorders. Moreover, whether their behaviour can adapt to developmental perturbations, generating robustness, has not been explored. Here, we show that Gli1+ cells are the fetal precursors of postnatal LLCPs. Moreover, while during normal growth they remain mostly dormant until postnatal stages, in response to cartilage-targeted cell-cycle arrest they increase their cartilage contribution, compensating for the perturbation. We further show that, during the response to challenge, some of the reparative Gli1+ cells originate from Pdgfra+ cells outside the cartilage anlage. Elucidating how stromal cells become Gli1+ LLCPs could shed light on developmental robustness and lead to growth-boosting therapies.

developmental biology↗

bioRxiv2024

ACTN3 genotype influences androgen response in skeletal muscle

AbstractAndrogens are vital for the maintenance of muscle mass and their anabolic effects are primarily exerted through the androgen receptor (AR). Accumulating evidence in humans and mice suggests that circulating androgens, AR and androgen response are influenced by ACTN3 (- actinin-3), also known as "the gene for speed". One in 5 people worldwide are -actinin-3 deficient due to homozygous inheritance of a common null polymorphism (577X) in ACTN3. In this study, we show that -actinin-3 deficiency decreases baseline AR in skeletal muscles of mice and humans, in both males and females, and that AR expression directly correlates with ACTN3 in a dosage dependent manner. We further demonstrate in Actn3 knockout mice that - actinin-3 deficiency increases muscle wasting induced by androgen deprivation and reduces the muscle hypertrophic response to dihydrotestosterone and this is mediated by differential activation of pathways regulating amino acid metabolism, intracellular transport, MAPK signalling, autophagy, mitochondrial activity and calcineurin signalling. Gene set enrichment and protein analyses indicate that the absence of -actinin-3 results in a failure to coactivate many of these pathways in response to changes in androgens, and relies on leveraging mitochondrial remodelling and calcineurin signalling to restore muscle homeostasis. We further identified 7 genes that are androgen sensitive and -actinin-3-dependent in expression, and whose functions correspond to these processes. Our results highlight the pivotal role of - actinin-3 in various processes associated with the regulation of protein turnover and muscle mass, and suggest that ACTN3 genotype is a genetic modifier of androgen action in skeletal muscle.

genetics↗

bioRxiv2023

The WalKR two-component regulator coordinates cell wall homeostasis with DNA replication in Staphylococcus aureus

Among the 16 two-component systems (TCSs) in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodelling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including ChIP-seq, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS); translation (rplK); DNA compaction (hup); initiation of DNA replication (dnaA, hup); and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by co-ordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Collectively, our findings address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics.

microbiology↗

bioRxiv2021

An iTSC-derived placental model of SARS-CoV-2 infection unveils ACE2-dependent susceptibility in syncytiotrophoblasts

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has caused a global health crisis. The primary site of infection is in the respiratory tract but the virus has been associated with a variety of complications involving the gastrointestinal and cardiovascular systems. Since the virus affects a variety of tissue types, there has been interest in understanding SARS-CoV-2 infection in early development and the placenta. ACE2 and TMPRSS2, two genes that are critical for SARS-CoV-2 virus entry are expressed in placenta-specific cell types including extravillous trophoblasts (EVTs) and especially, syncytiotrophoblasts (STs). The potential of SARS-CoV-2 to infect these placental cells and its effect on placental development and function is still unclear. Furthermore, it is crucial to understand the possible mechanism of vertical transmission of SARS-CoV-2 through the placenta. Here, we developed an in vitro model of SARS-CoV-2 infection of placental cell types using induced trophoblast stem cells (iTSCs). This model allowed us to show that STs but not EVTs are infected. Importantly, infected STs lack the expression of key differentiation genes, lack typically observed differentiated morphology and produce significantly lower human chorionic gonadotropin (HCG) compared to non-infected controls. We also show that an anti-ACE2 antibody prevents SARS-CoV-2 infection and restores normal ST differentiation and function. We highlight the establishment of a platform to study SARS-CoV-2 infection in early placental cell types, which will facilitate investigation of antiviral therapy to protect the placenta during early pregnancy and development.

cell biology↗

bioRxiv2020

Macrophages provide a transient muscle stem cell niche via NAMPT secretion

Skeletal muscle is paradigmatic of a regenerative tissue that repairs itself via the activation of a resident stem cell1. Termed the satellite cell, these normally quiescent cells are induced to proliferate by ill-defined wound-derived signals2. Identifying the source and nature of these pro-regenerative cues has been hampered by an inability to visualise the complex cellular interactions that occur within the wound environment. We therefore developed a zebrafish muscle injury model to systematically capture satellite cell interactions within the injury site, in real time, throughout the repair process. This analysis identified that a specific subset of macrophages dwells within the injury, establishing a transient but obligate stem cell niche required for stem cell proliferation. Single cell profiling identified specific signals secreted from dwelling macrophages that include the cytokine, Nicotinamide phosphoribosyltransferase (NAMPT/Visfatin/PBEF). Here we show that NAMPT secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (CCR5) expressed on muscle stem cells. This analysis reveals that along with their well-described ability to modulate the pro-inflammatory and anti-inflammatory phases of wound repair, specific macrophage populations also provide a transient stem cell-activating niche, directly supplying pro-proliferative cues that govern the timing and rate of muscle stem cell-mediated repair processes.

cell biology↗

bioRxiv2020

Transcriptome analysis of the zebrafish atoh7-/- mutant, lakritz, highlights Atoh7-dependent genetic networks with potential implications for human eye diseases

Expression of the bHLH transcription protein Atoh7 is a crucial factor conferring competence to retinal progenitor cells for the development of retinal ganglion cells. A number of studies have emerged establishing ATOH7 as a retinal disease gene. Remarkably, such studies uncovered ATOH7 variants associated with global eye defects including optic nerve hypoplasia, microphthalmia, retinal vascular disorders and glaucoma. The complex genetic networks and cellular decisions arising downstream of atoh7 expression, and how their dysregulation cause development of such disease traits remains unknown. To begin to understand such Atoh7-dependent events in vivo we performed transcriptome analysis of wild type and atoh7 mutant (lakritz) zebrafish embryos at the onset of retinal ganglion cell differentiation. We investigated in silico interplays of atoh7 and other disease-related genes and pathways. By network reconstruction analysis of differentially expressed genes we identified gene clusters enriched in retinal development, cell cycle, chromatin remodelling, stress response and Wnt pathways. By weighted gene coexpression network we identified coexpression modules affected by the mutation and enriched in retina development genes tightly connected to atoh7. We established the groundwork whereby Atoh7-linked cellular and molecular processes can be investigated in the dynamic multi-tissue environment of the developing normal and diseased vertebrate eye.

developmental biology↗

bioRxiv2020

TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novel Nanog regulators in mouse embryonic stem cells

Cellular identity is ultimately controlled by transcription factors (TFs), which bind to specific regulatory elements (REs) within the genome to regulate gene expression and cell fate changes. While recent advances in genome-wide epigenetic profiling techniques have significantly increased our understanding of which REs are utilized in which cell type, it remains largely unknown which TFs and cofactors interact with these REs to modulate gene expression. A major hurdle in dissecting the whole composition of a multi-protein complex formed at a specific RE is the shortage of appropriate techniques. We have developed a novel method termed TALE-mediated Isolation of Nuclear Chromatin (TINC). TINC utilizes epitope-tagged TALEs to isolate a specific genomic region from the mammalian genome and includes a nuclei isolation and chromatin enrichment step for increased specificity. Upon cross-linking of the cells and isolation of the chromatin, the target region is purified based on affinity purification of the TALE and associated nucleic acid and protein molecules can be subjected to further analyses. A key TF in the pluripotency network and therefore in embryonic stem cells (ESCs) is NANOG. It is currently not fully understood how Nanog expression is regulated and consequently it remains unclear how the ESC state is maintained. Using TINC we dissected the protein complex formed at the Nanog promoter in mouse ESCs and identified many known and numerous novel factors.

genomics↗

bioRxiv2019

Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α

The important role of microglia, the brains resident immune cells, in Alzheimers disease (AD) is now well recognized, however their molecular and functional diversity and underlying mechanisms still remain controversial. To transcriptionally and functionally characterize the diversity of microglia in AD and aging, we isolated the amyloid plaque-containing (XO4+) and non-containing (XO4-) microglia from an AD mouse model. Transcriptomics analysis unveiled independent transcriptional trajectories in ageing and AD. XO4+ microglial transcriptomes linked plaque phagocytosis to altered [Formula] expression of bona fide late onset AD genetic risk factors. We further revealed that the XO4+ transcriptional program is present in a subset of human microglia from AD patients and is a direct and reversible consequence of A{beta} plaque phagocytosis. Conversely, XO4- microglia in AD displayed an accelerated ageing signature and contained more intracellular post synaptic material than plaque-containing microglia, despite reduced active synaptosome phagocytosis. Mechanistically, we predicted HIF1 as a core regulator of the XO4-/XO4+ axis, and further validated the mechanism in vitro using human stem cell-derived microglia like cells and primary human microglia. Together these findings unveiled the molecular mechanism underpinning the functional diversity of microglia in AD, providing opportunities to develop treatments targeted at subset specific manipulation of the microglial niche.

neuroscience↗