Biology subjects

Villa, C. E.

Publications and source records attributed to Villa, C. E..

11 recordsLinked to original sources

bioRxiv2025

A molecular cell atlas of endocrine signalling in human neural organoids

Hormonal signalling shapes the development of the human brain and its disruption is implicated in various neuropsychiatric conditions. However, a comprehensive and mechanistic understanding of how hormonal pathways orchestrate human neurodevelopment remains elusive. Here we present a multi-scale high resolution atlas of endocrine signalling in human neural organoids through systematic perturbations with agonists and inhibitors of seven key hormonal pathways: androgen (AND), estrogen (EST), glucocorticoid (GC), thyroid (THY), retinoic acid (RA), liver X (LX), and aryl hydrocarbon (AH). By integrating bulk and single-cell transcriptomics, high-throughput imaging and targeted steroidomics, we mapped the molecular and cellular consequences of their physiologically relevant perturbations. Retinoic acid exerted the most profound effect, promoting neuronal differentiation and maturation, consistent with its established role as a patterning factor. Our analysis further benchmarked neural organoids for in vitro endocrinology and neurotoxicology by confirming previously reported in vivo effects, such as induction of mTOR signalling by AND, alteration of disease relevant genes by GC and enhanced differentiation by TH. Furthermore, we observed that LX activation upregulates genes involved in cholesterol metabolism while AH inhibition promotes neuronal differentiation. We next uncovered extensive crosstalks between these endocrine pathways, as in the paradigmatic convergence induced by AND agonist and inhibitors of GC, TH, and LX, affecting genes related to protein folding and metabolic regulation, as also highlighted by weighted gene co-expression network analysis. Single-cell analyses pinpointed cell-type-specific responses to hormonal challenges, such as the caudalization of progenitors and neurons upon RA activation and the depletion of specific neurodevelopmental states upon AH activation. Finally, we dissected the cytoarchitectural and morphometric impact of hormonal perturbations and demonstrated that neural organoids possess active steroidogenic pathways that are functionally modulated by the tested compounds. This atlas provides a systematic quantification of the hormonal impact on human neurodevelopment, enabling the investigation of uncharted aspects in the developmental origins of neuropsychiatric traits. Through the empowering architecture of its knowledge base for iterative adoption by the community, this resource will thus be key to probe how environmental factors and genetic endocrine vulnerabilities contribute to neurodevelopmental outcomes, as well as to train advanced generative models for improving their predictive power on gene environment interactions in human neurodevelopment.

molecular biology↗

bioRxiv2025

Mapping and reprogramming microenvironment-induced cell states in human disease using generative AI

Tissue microenvironments reprogram local cellular states in disease, yet current computational spatial methods remain descriptive and do not simulate tissue perturbation. We present MintFlow, a generative AI algorithm that learns how the tissue microenvironment influences cell states and predicts how tissue perturbations can reprogram them. Applied to three human diseases, MintFlow uncovered distinct pathogenic spatial reprogramming in inflammatory and tumor microenvironments. In atopic dermatitis, MintFlow identified a novel, spatially-imprinted, type 2 (IL13+ITGAE+) epidermal T resident memory cell population (type 2 TRM), and decoded signaling pathways within the perivascular lymphoid niche. In melanoma, MintFlow identified fibrotic stroma resembling keloid scar tissue. In kidney cancer, MintFlow resolved immunosuppressed CD8+ T cell states within tertiary lymphoid structures. Furthermore, MintFlow enabled in silico perturbations of disease-relevant cell states and tissue environments. Regulatory T cell modulation in atopic dermatitis was predicted to suppress the pro-inflammatory tissue environment, supporting manipulation of these cells as a therapeutic target. In kidney cancer, in silico T cell replacement recapitulated immune checkpoint blockade, while spatially targeted macrophage depletion reverted immunosuppressed T cell states. The corresponding gene programs correlated with survival in large kidney cancer patient cohorts. Together, these findings position MintFlow as a tool for unbiased disease mechanism prediction and in silico perturbation, accelerating translational hypothesis generation and guiding therapeutic strategies.

genomics↗

bioRxiv2025

Regulatory logic of human cortex evolution by combinatorial perturbations

Comparative genomic studies between contemporary and extinct hominins revealed key evolutionary modifications, but their number has hampered a system level investigation of their combined roles in scaffolding modern traits. Through multi-layered integration we selected 15 genes carrying nearly fixed sapiens-specific protein-coding mutations and developed a scalable design of combinatorial CRISPR-Cas9 bidirectional perturbations to uncover their regulatory hierarchy in cortical brain organoids. Interrogating the effects of overexpression and downregulation for all gene pairs in all possible combinations, we defined their impact on transcription and differentiation and reconstructed their regulatory architecture. We uncovered marked cell type-specific effects, including the promotion of alternative fates and the emergence of interneuron populations, alongside a core subnetwork comprising KIF15, NOVA1, RB1CC1 and SPAG5 acting as central regulator across cortical cell types.

systems biology↗

bioRxiv2025

A harmonized ovarian cancer scRNA-seq atlas to dissect disease heterogeneity underlying metastatization and chemoresistance

The advent of single cell technology has enabled researchers with the ability to achieve unprecedented resolution in the characterization of biological systems. The consequent increasing availability of single cell dataset brought about the possibility to obtain comprehensive reference atlases, to shed light on the molecular and cellular foundations of healthy and diseased tissues. This process has highlighted the need to integrate datasets from different sources while being able to distinguish true biological signal from technical confounders. While this issue is widespread to most biological settings, it holds especially true for cancer samples, which are characterized by a diffused inter- and intra-patient phenotypic heterogeneity. To address this issue, here we developed a novel integration method tailored to highly heterogeneous single cell transcriptomic data and applied it to one of the quintessential heterogeneous cancer type, namely high-grade serous ovarian cancer, to generate the first reference atlas for this disease. By identifying patient-specific cell populations and deriving metacells, we were able to preserve inter-patient biological variability. Using a variational autoencoder, we integrated metacell data, revealing an evolving landscape of cell states along disease progression and treatment for each of the main cell types constituting the dataset. Also, we showed the potential of this resource by identifying diffused and tissue/treatment-specific cell-to-cell interactions. Finally, the generated integration model allowed to expand the atlas with additional data, granting iterative refinement over time of this disease reference. Our strategy now provides a valuable resource for the cancer research community, facilitating the investigation of tumor heterogeneity towards the development of novel therapeutic strategies.

cancer biology↗

bioRxiv2023

Multiplexing cortical brain organoids for the longitudinal dissection of developmental traits at single cell resolution

The combination of brain organoid and single cell omic technologies holds transformative potential to dissect human neurobiology at high resolution and with mechanistic precision. Delivering this promise in the context of human neurodiversity, physiological and pathological alike, requires however a major leap in scalability, given the need for experimental designs that include multiple individuals and, prospectively, population cohorts. To lay the foundation for this, we implemented and benchmarked complementary strategies to multiplex brain organoids. Following an extended longitudinal design with a uniquely informative set of timepoints, we pooled cells from different induced pluripotent stem cell lines either during organoids generation (upstream multiplexing in mosaic models) or before single cell-RNAseq library preparation (downstream multiplexing). We developed a new method, SCanSNP, and an aggregated call to deconvolve organoids cell identities, overcoming current criticalities in doublets prediction and low quality cells identification and improving accuracy over state of the art algorithms. Integrating single cell transcriptomes and analysing cell types across neurodevelopmental stages and multiplexing modalities, we validated the feasibility of both multiplexing methods in charting neurodevelopmental trajectories at high resolution, linking their specificity to genetic variation between individual lines. Together, this multiplexing suite of experimental and computational methods provides an enabling resource for disease modelling at scale and paves the way towards an in vitro epidemiology paradigm.

neuroscience↗

bioRxiv2022

GTF2I dosage regulates neuronal differentiation and social behavior in 7q11.23 neurodevelopmental disorders

Copy number variations at 7q11.23 cause neurodevelopmental disorders with shared and opposite manifestations. Deletion causes Williams-Beuren syndrome (WBS), while duplication causes 7q11.23 microduplication syndrome (7Dup). Converging evidence indicates GTF2I, from the 7q11.23 locus, is a key mediator of the cognitive-behavioral phenotypes associated with WBS and 7Dup. Here we integrate molecular profiling of patient-derived cortical organoids (COs) and transgenic mouse models to dissect 7q11.23 disease mechanisms. Proteomic and transcriptomic profiling of COs revealed opposite dynamics of neural progenitor proliferation and transcriptional imbalances, leading to precocious excitatory neuron production in 7Dup. The accelerated excitatory neuron production in 7Dup COs could be rescued by GTF2I knockdown. Transgenic mice with Gtf2i duplication recapitulated early neuronal differentiation defects and ASD-like behaviors. Remarkably, inhibition of LSD1, a downstream effector of GTF2I, was sufficient to rescue ASD-like phenotypes. We propose that the GTF2I-LSD1 axis constitutes a molecular pathway amenable to therapeutic intervention.

neuroscience↗

bioRxiv2022

RAGE engagement by SARS-CoV-2 enables monocyte infection and underlies COVID-19 severity

The spread of SARS-CoV-2 has fueled the COVID-19 pandemic with its enduring medical and socioeconomic challenges due to subsequent waves and long-term consequences of great concern. Here we charted the molecular basis of COVID-19 pathogenesis, by analysing patients immune response at single-cell resolution across disease course and severity. This approach uncovered cell subpopulation-specific dysregulation in COVID-19 across disease course and severity and identified a severity-associated activation of the receptor for advanced glycation endproduct (RAGE) pathway in monocytes. In vitro experiments confirmed that monocytes bind the SARS-CoV-2 S1-RBD via RAGE and that RAGE-Spike interactions drive monocyte infection. Our results demonstrate that RAGE is a novel functional receptor of SARS-CoV-2 contributing to COVID-19 severity. One-Sentence SummaryMonocyte SARS-CoV-2 infection via the receptor for advanced glycation endproduct triggers severe COVID-19.

immunology↗

bioRxiv2020

CHD8 haploinsufficiency alters the developmental trajectories of human excitatory and inhibitory neurons linking autism phenotypes with transient cellular defects

Chromodomain helicase DNA-binding 8 (CHD8) is one of the most frequently mutated genes causative of autism spectrum disorder (ASD). While its phenotypic spectrum often encompasses macrocephaly and hence implicates cortical abnormalities in this form of ASD, the neurodevelopmental impact of human CHD8 haploinsufficiency remains unexplored. Here we combined human cerebral organoids and single cell transcriptomics to define the effect of ASD-linked CHD8 mutations on human cortical development. We found that CHD8 haploinsufficiency causes a major disruption of neurodevelopmental trajectories with an accelerated generation of inhibitory neurons and a delayed production of excitatory neurons alongside the ensuing protraction of the proliferation phase. This imbalance leads to a significant enlargement of cerebral organoids aligned to the macrocephaly observed in patients with CHD8 mutations. By adopting an isogenic design of patient-specific mutations and mosaic cerebral organoids, we define genotype-phenotype relationships and uncover their cell-autonomous nature. Finally, our results assign different CHD8-dependent molecular defects to particular cell types, pointing to an abnormal and extended program of proliferation and alternative splicing specifically affected in, respectively, the radial glial and immature neuronal compartments. By identifying temporally restricted cell-type specific effects of human CHD8 mutations, our study uncovers developmental alterations as reproducible endophenotypes for neurodevelopmental disease modelling.

neuroscience↗

bioRxiv2018

Longitudinal dissection in brain organoids at single cell resolution uncovers the developmental role of GSK3 in human corticogenesis

The regulation of proliferation and polarity of neural progenitors is crucial for the development of the brain cortex, with modes and timings of cell division intimately related to the stereotypical acquisition of layer-specific neuronal identities. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remain to be elucidated. Here we harness human cortical brain organoids to probe, at single cell resolution, the longitudinal impact of GSK3 inhibition through multiple developmental stages. Our results indicate that chronic GSK3 inhibition increases the proliferation of neural progenitors and causes massive derangement of cortical tissue architecture. Surprisingly, single cell transcriptome profiling revealed only a discrete impact on early neurogenesis and uncovered the outer radial glia and the astrogenic lineage as the main GSK3-dependent developmental domains. Through this first single cell-level dissection of the GSK3 regulatory network in human corticogenesis, our work uncovers a remarkably specific conduit between the architecture of progenitor niches and lineage specification.

developmental biology↗

bioRxiv2018

A cell-of-origin epigenetic tracer reveals clinically distinct subtypes of high grade serous ovarian cancer

High grade serous ovarian cancer (HGSOC) is a major unmet need in oncology. The persistent uncertainty on its originating tissue has contributed to hamper the discovery of oncogenic pathways and effective therapies. Here we define the DNA methylation print that distinguishes the human fimbrial (FI) and ovarian surface epithelia (OSE) and develop a robust epigenetic cell-of-origin tracer that stratifies HGSOC in FI-and OSE-originated tumors across all available cohorts. We translate this origin-based stratification into a clinically actionable transcriptomic signature, demonstrating its prognostic impact on patients survival and identifying novel network level dysregulations specific for the two disease subtypes.

cancer biology↗

bioRxiv2018

Single cell derived organoids capture the self-renewing subpopulations of metastatic ovarian cancer

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients ascites, establishing the conditions for propagating them as single-cell derived ovarian cancer organoids (scOCOs). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that scOCOs retain and amplify key subpopulations from the original patients samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method transforms the prospects of precision oncology for ovarian cancer.

cancer biology↗