Biology subjects

Garcia-Alonso, L.

Publications and source records attributed to Garcia-Alonso, L..

13 recordsLinked to original sources

bioRxiv2026

Full-thickness spatial transcriptomics of the human uterus reveals basalis niche architecture and regeneration gradients during menstrual breakdown

The human endometrium (uterine lining) undergoes cyclical breakdown and scarless regeneration during each menstrual cycle, representing an exceptional model of adult tissue renewal. Regeneration is driven primarily by progenitor cells retained within the deep, basalis compartment during menstruation, yet the full-depth spatiotemporal dynamics of this process have remained understudied due to anatomical and technical limitations. Here, we map spatial gene-expression gradients across the full thickness of the human endometrium, from the myometrial-endometrial boundary to the luminal surface, using high-resolution spatial transcriptomics integrated with single-cell transcriptomics. We profile more than ten million cells from biopsies, hysterectomy samples and menstrual fluid, enriching for the menstrual and proliferative phases, which are underrepresented in previous studies. We show that endometrial breakdown, regeneration and rapid luminal re-epithelialisation are concurrent rather than temporally separated, organised across distinct tissue compartments, revealing a mode of tissue renewal in which shedding and repair operate simultaneously. Continuous basalis-to-luminal transcriptional gradients link progenitor identity, niche signalling, and tissue remodelling, defining a coordinated regenerative axis spanning the full tissue depth. We resolve the basalis epithelial niche at unprecedented molecular resolution, identifying for the first time a discrete, predominantly quiescent progenitor-like epithelial subset and specialised supporting SFRP5+ fibroblasts, both characterised by WNT inhibition, alongside lymphoid aggregates, forming a multi-component architecture that persists after menopause, consistent with a long-lived regenerative reservoir. Together, these findings establish spatial transcriptional gradients as a central organising principle of endometrial renewal, providing a molecular framework for understanding disorders of menstruation, implantation failure, and impaired tissue repair.

cell biology↗

bioRxiv2026

Spatial atlas of the ovary identifies molecular events in primordial follicle activation in humans

The human ovarian reserve is established prenatally, when granulosa cells encapsulate germ cells to form a pool of quiescent primordial follicles, that ultimately determines the female reproductive and endocrine lifespan. From birth to menopause, subsets of these quiescent follicles, located in the thin outer ovarian cortex, are activated and undergo a growth programme with progressive inward migration to the inner cortex, before either undergoing atresia or, during reproductive age, ovulation. Disruption of this process may lead to infertility, metabolic disorders and early menopause, yet early follicle development remains largely poorly understood. Here, we generate the most comprehensive single-cell and spatial multiomics atlas of the human pre- and postnatal ovarian cortex, integrating transcriptomic and chromatin profiles from over four million cells obtained from fetal and newly profiled pediatric and adult donors. We resolve the early granulosa cell trajectory at unprecedented resolution and identify a retinoic acid-associated regulatory switch accompanying follicle activation. We further show that stromal fibroblasts are not homogeneous, but instead form a dynamic scaffold establishing previously unrecognised morphogen and paracrine gradients that organise the cortex into functional niches supporting quiescent, growing, and atretic follicles. Finally, we identify ovarian lipid associated macrophages (oLAMs) that localise around follicles and are likely to support tissue remodelling during folliculogenesis. Together, this atlas provides a foundational blueprint for human ovarian development and homeostasis, and provides a framework for improving strategies in fertility preservation and in vitro follicle maturation.

cell biology↗

bioRxiv2025

Decoding human B cell ontogeny in prenatal and adult bone marrow and in vitro models via single-cell multiomics

In humans, the bone marrow becomes the primary site for B lymphopoiesis during the second trimester of pregnancy and continues throughout life. Prenatal and adult B cell progenitors play distinct roles in the aetiology and pathology of paediatric and adult hematopoietic malignancies, though the molecular drivers of these differences remain unclear. Here, we created a comprehensive multiomics atlas of over 500k cells covering immune and stromal compartment from prenatal and adult bone marrow, enabling high-resolution analysis of the cell-intrinsic and cell-extrinsic processes that modulate prenatal and adult B lymphopoiesis. Even though B cells follow broadly similar developmental trajectories, we identify a novel postnatal lateCLP subset equivalent to prenatal preProB cells, and uncover prenatal cells carry several signatures characteristic of leukemias, including enhanced proliferation, higher RAG1/RAG2 activity, extrinsic B cell signals such as IL7, and lower retention signals in the bone marrow. We also developed and characterised, at both cellular and molecular levels, a new experimental framework for generating B cell precursors from human induced pluripotent stem cells (hiPSCs), and show that it faithfully recapitulates key stages of B cell differentiation. Together, our single-cell multiomics atlas of B lymphopoiesis in vivo and in vitro offers detailed insights into the unique molecular features of prenatal and adult B cell lymphopoiesis, and serves as a powerful resource for investigating the early events that contribute to haematological disorders.

immunology↗

bioRxiv2025

Atlas-scale metabolic activities inferred from single-cell and spatial transcriptomics

Metabolism supplies energy, building blocks, and signaling molecules vital for cell function and communication, but methods to directly measure it at single-cell and/or spatial resolutions remain technically challenging and inaccessible for most researchers. Single-cell and spatial transcriptomics offer high-throughput data alternatives with a rich ecosystem of computational tools. Here, we present scCellFie, a computational framework to infer metabolic activities from human and mouse transcriptomic data at single-cell and spatial resolution. Applied to [~]30 million cell profiles, we generated a comprehensive metabolic atlas across human organs, identifying organ- and cell-type-specific activities. In the endometrium, scCellFie reveals metabolic programs contributing to healthy tissue remodeling during the menstrual cycle, with temporal patterns replicated in data from in vitro cultures. We also uncover disease-associated metabolic alterations in endometriosis and endometrial carcinoma, linked to proinflammatory macrophages, and metabolite-mediated epithelial cell communication, respectively. Ultimately, scCellFie provides a scalable toolbox for extracting interpretable metabolic functionalities from transcriptomic data.

bioinformatics↗

bioRxiv2024

Spatiotemporal map of the developing human reproductive tract at single-cell resolution

The human reproductive tract plays an essential role in species perpetuation. Its development involves complex processes of sex specification, tissue patterning and morphogenesis, which, if disrupted, can cause lifelong health issues, including infertility. Here, we generated an extensive single-cell and spatial multi-omic atlas of the human reproductive tract during prenatal development, which allowed us to answer questions that smaller-scale, organ-focused experiments could not address before. We identified potential regulators of sexual dimorphism in reproductive organs, pinpointing novel genes involved in urethral canalisation of the penis, with relevance to hypospadias. By combining histological features with gene expression data, we defined the transcription factors and cell signalling events required for the regionalisation of the Mullerian and Wolffian ducts. This led to a refinement of how the HOX code is established in the distinct reproductive organs, including increased expression of thoracic HOX genes in the rostral mesenchyme of the fallopian tube and epididymis. Our study further revealed that the epithelial regionalisation of the fallopian tube and epididymis required for sperm maturation in adulthood is established early in development. In contrast, later events in gestation or postnatally are necessary for the regionalisation of the uterocervical canal epithelium. By mapping sex-specific reproductive tract regionalisation and differentiation at the cellular level, our study offers valuable insights into the causes and potential treatments of reproductive disorders.

developmental biology↗

bioRxiv2023

An integrated single-cell reference atlas of the human endometrium

The human endometrium, the inner lining of the uterus, exhibits complex, dynamic changes throughout the menstrual cycle in response to ovarian hormones. Aberrant response of endometrial cells to hormones is associated with multiple disorders, including endometriosis. Previous single-cell studies of the endometrium profiled a limited number of donors and lacked consensus in defining cell types and states. Here, we introduce the Human Endometrial Cell Atlas (HECA), a high-resolution single-cell reference atlas, combining published and newly generated single-cell transcriptomics datasets of endometrial biopsies of women with and without endometriosis. The HECA assigned consensus cell types and states, and uncovered novel ones, which we mapped in situ using spatial transcriptomics. We quantified how coordinated interactions between cell states in space and time contribute to endometrial regeneration and differentiation. In the continuously changing functionalis layer, we identified an intricate coordination of TGF{beta} signalling between stromal and epithelial cells, likely crucial for cell differentiation. In the basalis layer, we defined signalling between fibroblasts and a new epithelial cell population expressing epithelial stem/progenitor markers, suggesting their role in endometrial regeneration. Additionally, integrating the HECA single-cell data with genome-wide association study data and comparing endometrial samples from women with and without endometriosis, we pinpointed subsets of decidualised stromal cells and macrophages as the most dysregulated cell states in endometriosis. Overall, the HECA is an invaluable resource for studying endometrial physiology, investigating endometrial disorders, and guiding the creation of endometrial microphysiological in vitro systems.

genomics↗

bioRxiv2022

Spatially resolved single-cell multiomics map of human trophoblast differentiation in early pregnancy

The relationship between the human placenta, the extraembryonic organ built by the fetus, and the decidua, the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) anchor the placenta and infiltrate the decidua, transforming the maternal arteries into high conductance vessels. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia. Despite its importance, how EVT invasion is regulated in humans is still unclear due the inaccessibility of the entire pregnant uterus and, until recently, a lack of reliable in vitro models. Here, we have generated a spatially-resolved multiomics single-cell atlas of the entire maternal-fetal interface including the myometrium, allowing us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to elucidate the main regulatory programmes mediating EVT invasion and show that they are preserved in trophoblast organoids. We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We reconstruct the cell-cell communication events contributing to trophoblast invasion and GC formation, and define the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provides a comprehensive analysis of postimplantation trophoblast differentiation in humans that can be used as a blueprint to design accurate multilineage placental in vitro models.

genomics↗

bioRxiv2021

Single-Cell Atlas of Common Variable Immunodeficiency reveals germinal center-associated epigenetic dysregulation in B cell responses

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, is characterized by impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and a wide range of phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. We used single-cell epigenomics and transcriptomics to create a cell census of naive-to-memory B cell differentiation in a pair of CVID-discordant MZ twins. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B cells that mirror defective cell-cell communication defects following activation. These findings were validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naive-to-memory B-cell transition in CVID and reveal links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, paves the way for future diagnosis and treatments of CVID patients.

immunology↗

bioRxiv2021

Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated single-cell and spatial reference maps of the human uterus and 3D endometrial organoid cultures. We dissect the signalling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids highlights common pathways regulating the differentiation of secretory and ciliated lineage in vivo and in vitro. We show in vitro that downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. These mechanistic insights provide a platform for future development of treatments for a range of common endometrial disorders including endometriosis and carcinoma.

genomics↗

bioRxiv2019

The functional landscape of the human phosphoproteome

Protein phosphorylation is a key post-translational modification regulating protein function in almost all cellular processes. While tens of thousands of phosphorylation sites have been identified in human cells to date, the extent and functional importance of the phosphoproteome remains largely unknown. Here, we have analyzed 6,801 publicly available phospho-enriched mass spectrometry proteomics experiments, creating a state-of-the-art phosphoproteome containing 119,809 human phosphosites. To prioritize functional sites, 59 features indicative of proteomic, structural, regulatory or evolutionary relevance were integrated into a single functional score using machine learning. We demonstrate how this prioritization identifies regulatory phosphosites across different molecular mechanisms and pinpoint genetic susceptibilities at a genomic scale. Several novel regulatory phosphosites were experimentally validated including a role in neuronal differentiation for phosphosites present in the SWI/SNF SMARCC2 complex member. The scored reference phosphoproteome and its annotations identify the most relevant phosphorylations for a given process or disease addressing a major bottleneck in cell signaling studies.

genomics↗

bioRxiv2018

Benchmark and integration of resources for the estimation of human transcription factor activities

Prediction of transcription factor (TF) activities from the gene expression of their targets (i.e. TF regulon) is becoming a widely-used approach to characterize the functional status of transcriptional regulatory circuits. Several strategies and datasets have been proposed to link the target genes likely regulated by a TF, each one providing a different level of evidence. The most established ones are: (i) manually curated repositories, (ii) interactions derived from ChIP-seq binding data, (iii) in silico prediction of TF binding on gene promoters, and (iv) reverse-engineered regulons from large gene expression datasets. However, it is not known how these different sources of regulons affect the TF activity estimations, and thereby downstream analysis and interpretation. Here we compared the accuracy and biases of these strategies to define human TF regulons by means of their ability to predict changes in TF activities in three reference benchmark datasets. We assembled a collection of TF-target interactions among 1,541 TFs, and evaluated how the different molecular and regulatory properties of the TFs, such as the DNA-binding domain, specificities or mode of interaction with the chromatin, affect the predictions of TF activity changes. We assessed their coverage and found little overlap on the regulons derived from each strategy and better performance by literature-curated information followed by ChIP-seq data. We provide an integrated resource of all TF-target interactions derived through these strategies with a confidence score, as a resource for enhanced prediction of TF activities.

bioinformatics↗

bioRxiv2017

Transcription factor activities enhance markers of drug response in cancer

Transcriptional dysregulation is a key feature of cancer. Transcription factors (TFs) are the main link between signalling pathways and the transcriptional regulatory machinery of the cell, positioning them as key oncogenic inductors and therefore potential targets of therapeutic intervention. We implemented a computational pipeline to infer TF regulatory activities from basal gene expression and applied it to publicly available and newly generated RNA-seq data from a collection of 1,010 cancer cell lines and 9,250 primary tumors. We show that the predicted TF activities recapitulate known mechanisms of transcriptional dysregulation in cancer and dissect mutant-specific effects in driver genes. Importantly, we show the potential for predicted TF activities to be used as markers of sensitivity to the inhibition of their upstream regulators. Furthermore, combining these inferred activities with existing pharmacogenomic markers significantly improves the stratification of sensitive and resistant cell lines for several compounds. Our approach provides a framework to link driver genomic alterations with transcriptional dysregulation that helps to predict drug sensitivity in cancer and to dissect its mechanistic determinants.

cancer biology↗

bioRxiv2016

Chromosomal rearrangements are commonly post-transcriptionally attenuated in cancer

Chromosomal rearrangements, despite being detrimental, are ubiquitous in cancer and often act as driver events. The effect of copy number variations (CNVs) on the cellular proteome of tumours is poorly understood. Therefore, we have analysed recently generated proteogenomic data-sets on 282 tumour samples to investigate the impact of CNVs in the proteome of these cells. We found that CNVs are post-transcriptionally attenuated in 23-33% of proteins with an enrichment for protein complexes. Complex subunits are highly co-regulated and some act as rate-limiting steps of complex assembly, indirectly controlling the abundance of other complex members. We identified 48 such regulatory interactions and experimentally validated AP3B1 and GTF2E2 as controlling subunits. Lastly, we found that a gene-signature of protein attenuation is associated with increased resistance to chaperone and proteasome inhibitors. This study highlights the importance of post-transcriptional mechanisms in cancer which allow cells to cope with their altered genomes.

genomics↗