Biology subjects

Pellegrino, E.

Publications and source records attributed to Pellegrino, E..

8 recordsLinked to original sources

bioRxiv2026

The Genetic Discovery (GeDi) resource: Deep characterization of a multi-ancestry panel of human reference iPSCs for functional genomic studies

Human induced pluripotent stem cells (iPSCs) are essential tools for disease modelling, drug discovery, and functional genomics, yet the field remains anchored to a small number of reference lines, limiting our understanding of how genetic background shapes cellular phenotypes across human populations. Here we describe the Genetic Discovery iPSC panel (GeDi), eight clonal iPSC lines reprogrammed from male and female donors that broadly span global human population diversity. Ancestry analysis confirmed that each line's genetic profile is concordant with donor self-reported ancestry. All GeDi lines are pluripotent and passed comprehensive quality control, demonstrating Sendai virus clearance, intact p53 pathway integrity, reproducible proliferative capacity, and compatibility with standardized genome editing workflows. Transcriptome profiling across five differentiated lineages covered 80% of annotated protein-coding genes and revealed stable donor-intrinsic variability in gene expression within each lineage. Haplotype-resolved genome assemblies were used to catalog structural variants and regions of absence of heterozygosity invisible to short-read sequencing. Whole-genome methylation analysis confirmed successful epigenetic reprogramming, catalogued the status of imprinted loci and captured the early dynamics of erosion of X chromosome inactivation (XCI) in female lines. The GeDi panel is available from The Jackson Laboratory as a quality-controlled resource designed to support functional genomic studies across varied genetic backgrounds.

cell biology↗

bioRxiv2025

Autologous human iPSC-derived Alveolus-on-Chip reveals early pathological events of M. tuberculosis infection

Immunocompetent and experimentally accessible alveolar systems to study human respiratory diseases are lacking. Here, we developed a single donor human induced pluripotent stem cell (iPSC)-derived Lung-on-Chip (iLoC) containing Type II and I alveolar epithelial cells, vascular endothelial cells, and macrophages in a microfluidic device that mimic lung 3D mechanical stretching and air-liquid interface. Imaging and scRNA-seq analysis revealed that the iLoC recapitulated cellular profiles present in the human distal lung. Infection of the iLoC with the human pathogen Mycobacterium tuberculosis (Mtb) showed that both macrophages and epithelial cells were infected and showed limited bacterial replication. Stochastically, large macrophage clusters containing necrotic core-like structure and Mtb replication were observed. A genetically engineered autophagy deficient iLoC revealed that after Mtb infection, macrophage necrosis was higher upon ATG14 deficiency without bacterial replication. Altogether, we report an autologous, genetically tractable human alveolar model to study lung diseases and therapies.

bioengineering↗

bioRxiv2024

Near 100% genome editing efficiency with a one-step all RNA prime editing system in a human reporter iPSC line

CRISPR/Cas9 nucleases offer powerful tools for genome editing but can cause unintended mutations at the target site due to the creation of double-stranded DNA breaks. Prime editing (PE) is arguably a safer technology as it relies on the creation of single-strand DNA nicks and avoids possible indels. Despite its precision, current PE systems are limited by relatively low editing efficiency in non-immortalized cells. Here, we optimized prime editing in human induced pluripotent stem cells (hiPSCs) using a fluorescence-based assay to achieve near-complete editing of a BFP transgene. Using an all-RNA approach, precise prime editing of a single nucleotide variant was observed in >95% of cells with minimal unintended edits at the target site.

molecular biology↗

bioRxiv2024

ATG9A and ARFIP2 cooperate to regulate PI4P levels for lysosomal repair

Lysosome damage activates multiple pathways to prevent lysosome-dependent cell death, including a repair mechanism involving ER-lysosome membrane contact sites, phosphatidylinositol 4-kinase- 2a (PI4K2A), phosphatidylinositol-4 phosphate (PI4P) and oxysterol-binding protein-related proteins (ORPs), lipid transfer proteins. PI4K2A localizes to trans-Golgi network and endosomes yet how it is delivered to damaged lysosomes remains unknown. During acute sterile damage, and damage caused by intracellular bacteria, we show that ATG9A-containing vesicles perform a critical role in delivering PI4K2A to damaged lysosomes. ADP ribosylation factor interacting protein 2 (ARFIP2), a component of ATG9A vesicles, binds and sequesters PI4P on lysosomes, balancing ORP- dependent lipid transfer and promoting retrieval of ATG9A vesicles through recruitment of the adaptor protein complex-3 (AP-3). Our results reveal a role for mobilized ATG9A vesicles and ARFIP2 in lysosome homeostasis after damage and bacterial infection.

cell biology↗

bioRxiv2023

LRRK2 kinase dependent and independent function on endolysosomal repair promotes macrophage cell death

LRRK2 is commonly mutated in Parkinsons disease and has cell type-specific mechanisms of activation and function. In macrophages, LRRK2 is associated with lysosomes and is activated following lysosomal damage. However, effects of pathogenic LRRK2-G2019S in macrophages are unknown. Here, using primary mouse and human iPSC-derived macrophage (iPSDM) models of LRRK2-G2019S, we defined the substrates of LRRK2 after lysosomal damage. Using phosphoproteomics we found that LRRK2-G2019S and wild-type macrophages showed similar levels of Rab phosphorylation after lysosomal damage, with the exceptions of Rab12 and Rab35, which were increased and decreased, respectively, in LRRK2-G2019S. LRRK2-G2019S macrophages showed a LRRK2 kinase activity-independent deficit in lysosomal membrane repair which resulted in more cell death and increased apoptosis. Importantly, we recapitulated this phenotype in iPSDM from patients carrying the G2019S mutation, but not in isogenic control iPSDM. Altogether, we define here the signaling downstream of G2019S in macrophages and identify susceptibility to cell death after lysosomal damage as an important phenotype of this mutation.

cell biology↗

bioRxiv2022

Lysosomal damage drives mitochondrial proteome remodelling and reprograms macrophage immunometabolism

Transient lysosomal damage after infection with cytosolic pathogens or silica crystals uptake results in protease leakage. Whether limited leakage of lysosomal contents into the cytosol affects the function of cytoplasmic organelles is unknown. Here, we show that sterile and non-sterile lysosomal damage triggers a cell death independent proteolytic remodelling of the mitochondrial proteome in macrophages. Mitochondrial metabolic reprogramming required lysosomal leakage of Cathepsin B and Cathepsin L and was independent of proteasome degradation and mitophagy. In a mouse model of endomembrane damage, metabolic analysis confirmed that in vivo, live lung macrophages that internalised crystals displayed impaired mitochondrial function and increased glycolytic and lipid metabolism. Single-cell RNA-sequencing analysis of bronchoalveolar lavage revealed that lysosomal damage skewed metabolic and immune responses primarily in CD36+/LIPA+ and Krt79+/Car4+ subsets of alveolar macrophages. Importantly, modulation of macrophage metabolism with 2-Deoxy- d- glucose and oxamate impacted the host response to Mycobacterium tuberculosis (Mtb) infection in an endomembrane damage dependent manner. This work uncovers a new inter-organelle communication pathway, providing a general mechanism by which macrophages undergo mitochondrial metabolic reprograming after endomembrane damage.

cell biology↗

bioRxiv2022

Microcephaly-associated WDR62 mutations hamper Golgi apparatus-to-spindle pole shuttling in human neural progenitors

WDR62 is a spindle pole-associated scaffold protein with pleiotropic functions during corticogenesis. Recessive mutations in WDR62 are associated with structural brain abnormalities and account for the second most common cause of autosomal recessive primary microcephaly (MCPH), indicating WDR62 as a critical hub for human brain development. Here, we investigated a C-terminal truncating mutation (D955AfsX112) in WDR62 using induced pluripotent stem cells (iPSCs) obtained from a patient with MCPH2. We generated neuroepithelial stem (NES) cells and cerebro-cortical progenitors and neurons from patient-derived and isogenic retro-mutated iPSC lines. We found that WDR62 dysfunction resulted in impaired cell cycle progression and alterations of the neurogenic trajectories of iPSC neuroderivatives. Moreover, we report WDR62 localization at the Golgi apparatus during interphase, both in human neural progenitors in vitro and in human fetal brain tissue. WDR62 shuttling from the Golgi apparatus to spindle poles is dynamic and microtubule-dependent. Impairment of WDR62 function and localization results in severe neurodevelopmental abnormalities, thus delineating new mechanisms in MCPH etiology.

neuroscience↗

bioRxiv2021

Visualizing pyrazinamide action by live single cell imaging of phagosome acidification and Mycobacterium tuberculosis pH homeostasis

The intracellular population of Mycobacterium tuberculosis (Mtb) is dynamically segregated within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a dual-imaging approach that allows quantitative monitoring of host subcellular acidification and Mtb intrabacterial pH profiles by live-fluorescence microscopy in a biosafety level 3 laboratory. By combining this live imaging approach with pharmacological and genetic perturbations, we show that Mtb can maintain its intracellular pH independently of the surrounding pH in primary human macrophages. Importantly, we show that unlike bedaquiline (BDQ), isoniazid (INH) or rifampicin (RIF), the front-line drug pyrazinamide (PZA) displays antibacterial efficacy by acting as protonophore which disrupts intrabacterial pH homeostasis in cellulo. By using Mtb mutants with different intra-macrophage localisation, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this dual imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. HighlightsO_LIMtb maintains its intrabacterial pH inside both acidic and neutral subcellular microenvironments of human macrophages C_LIO_LIPyrazinamide, but not other frontline antibiotics, acts as a protonophore in cellulo C_LIO_LIPyrazinamide-mediated intrabacterial pH homeostasis disruption and antibacterial efficacy requires host endolysosomal acidification C_LIO_LICytosolic localisation mediated by ESX-1 contributes to pyrazinamide antibacterial activity resistance C_LIO_LIPyrazinamide conversion into pyrazinoic acid by the pyrazinamidase/nicotinamidase PncA is essential for its protonophore activity and efficacy in cellulo C_LI

microbiology↗