Biology subjects

Sordi, V.

Publications and source records attributed to Sordi, V..

6 recordsLinked to original sources

bioRxiv2025

Latent plasticity of the human pancreas across development, health, and disease.

The pancreas plays a central role in major human diseases, yet our understanding of its cellular diversity and plasticity remains incomplete. Here, we present a single-cell multiomics atlas of the human pancreas, profiling over four million cells and nuclei from 57 donors across fetal development, adult homeostasis, and type 2 diabetes (T2D). Integrating sc/snRNA-seq, snATAC-seq, VASA-seq, spatial transcriptomics (Xenium), and multiplexed proteomics (CODEX), we resolve gene expression, chromatin accessibility, and spatial organization at high resolution. We identify transcriptionally plastic centroacinar-like cells (pCACs) in adults with fetal-like features, delineate endocrine and exocrine lineage trajectories during development, and uncover HNF1A-defined beta cell epigenetic states. In T2D, we observe shifts in beta cell subtypes and altered regulatory programs. Glucose perturbation of healthy islets reveals cell-type-specific adaptation and stress responses. This atlas provides a foundational framework to understand pancreas biology and the role of cellular plasticity in regeneration and disease.

genomics↗

bioRxiv2025

Deep Learning for Predicting Stem Cell Efficiency for use in Beta Cell Differentiation

Recent clinical trial data have shown that cell therapy holds curative potential for type-1 diabetes, however the large amounts of lab-grown cells required is a substantial bottleneck. The necessary cell differentiation process exhibits substantial variability, even among clones of induced pluripotent stem cells generated from the same patient. In contrast to many established data sets in medical imaging, human experts struggle to see the difference between highly- and lowly-efficient cell clones. We therefore propose an image-based deep learning model to guide the selection of the most efficient stem cell clones. We apply different deep learning model architectures to learn the morphological differences between good and bad stem cell clones and classify them based on only phase-contrast imaging. To gain critical insight into the learned features and enhance trust in our model, we use layer-wise relevance propagation (LRP), and Fourier-based frequency analysis. Using an EfficientNet-V2-S model, we obtain a novel early prediction for the outcome of the differentiation process from patient-derived stem cell to {beta}-cells using only phase-contrast images. Clone level accuracy is 76.7 % at 24 hours and 96.7 % at 53 hours after start of differentiation. The LRP-generated attribution maps and structure factor analysis show that the structure of the cell population is an important predictive feature. All in all, we present a highly predictive model for successful stem cell differentiation from phase-contrast images, which learns biologically relevant features. This study is a proof-of-concept that deep learning combined with label-free imaging can guide selection of induced pluripotent stem cell clones, thereby reduce cost of {beta}-cell production, and bring curative treatments one step closer to the clinic.

bioinformatics↗

bioRxiv2024

Liraglutide treatment reverses unconventional cellular defects in induced pluripotent stem cell-derived β cells harboring a partially functional WFS1 variant

Aims/hypothesisWolfram Syndrome 1 (WS1) is a rare genetic disorder characterized by very heterogeneous clinical manifestations caused by variants of the WFS1 gene, which encodes for the Endoplasmic Reticulum (ER) protein Wolframin, involved in cellular stress response, Ca2+ handling and autophagy. Given the central role of Wolframin, elucidating the impact of WFS1 variants on cell functions is crucial to provide an association with clinical phenotypes. Therefore, as the understanding of patient-specific defects may also help to develop targeted therapeutic approaches, here we aimed at elucidating the impact on {beta} cell function of the c.316-1G>A mutation harboring the partially functional Wolframin that we have previously characterized, and the molecular changes following treatment with the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide. MethodsWe previously generated patient-derived iPSCs (WFS1) and isogenic line in which the c.316-1G>A mutation was genetically corrected (WFS1wt/757A>T), thus performed molecular analysis, including single cell RNAseq (scRNAseq), and functional studies on iPSC-derived {beta} cell (iBeta). Calcium flux imaging and dynamic perifusion assays were used to test glucose responsiveness of iBeta. Treatment with liraglutide was performed to investigate effects on glucose-stimulated insulin secretion (GSIS), unfolded protein response (UPR), autophagy and apoptosis. ResultsWe found that both WFS1 and WFS1wt/757A>T iBeta efficiently differentiated in vitro into pancreatic lineage, but WFS1 showed less mature endocrine phenotype, reduced glucose responsiveness and impaired insulin secretion compared to WFS1wt/757A>T counterpart. The Ca2+ dynamics were altered in WFS1 iBeta as Ca2+ oscillations after glucose challenge were not synchronized mainly due to the CACNA1D and SNAP25 downmodulation. Reduced insulin secretion was correlated with a decrease in PC1/3 levels and overall increase of RGS4 expression in WFS1 iBeta, whereas secretory defects correlated with accelerated autophagic flux. While the functional residual Wolframin in WFS1 iBeta controlled short-term ER stress, prolonged insults or inflammation highlighted ineffective UPR that make the cells unable to escape apoptosis. Interestingly, treatment with liraglutide restored the Ca2+ fluxes and secretory impairments, increasing glucose responsiveness and insulin release of WFS1 iBeta, while protecting these cells from cellular stress and inflammation-induced apoptosis. Conclusion/interpretationOur data highlighted alterations of key cellular pathways involved in WS1 {beta} cell maturation and GSIS and how the pharmacological targeting of the GLP-1/GLP-1R axis was able to restore the physiologic phenotype. This study points out the need to understand the patient-specific molecular determinants associated with WFS1 variants, to design effective therapies to treat the disease.

cell biology↗

bioRxiv2024

Implications of noncoding regulatory functions in the development of insulinomas

Insulinomas are rare neuroendocrine tumours arising from the pancreatic {beta}-cells. While retaining the ability to produce insulin, insulinomas feature aberrant proliferation and altered hormone secretion resulting in failure to maintain glucose homeostasis. With the aim of uncovering the role of noncoding regulatory regions and their aberrations to the development of these tumors, we coupled epigenetic and gene expression profiling with whole-genome sequencing. As a result, we mapped H3K27ac sites in the tumoral tissue and unraveled overlapping somatic mutations associated with changes in regulatory functions. Critically, these regions impact insulin secretion, tumor development and epigenetic modifying genes, including key components of the polycomb complex. Chromatin remodeling is apparent as insulinoma-selective regions are mostly clustered in regulatory domains, shared across patients and containing a specific set of regulatory sequences dominated by the binding motif of the transcription factor SOX17. Moreover, a large fraction of these regions are H3K27me3-repressed in unaffected {beta}-cells, suggesting that tumoral transition is coupled with derepression of {beta}-cell polycomb-targeted domains. Our work provides a compendium of aberrant cis-regulatory elements and transcription factors that alter {beta}-cell function and fate in their progression to pancreatic neuroendocrine tumors and a framework to identify coding and noncoding driver mutations.

genomics↗

bioRxiv2023

A WFS1 variant disrupting acceptor splice site uncovers the impact of alternative splicing on ER-stress independent β cell apoptosis in a patient with Wolfram syndrome.

Aims/hypothesisWolfram Syndrome 1 (WS1) is an inherited condition mainly manifesting in childhood-onset diabetes mellitus and progressive optic nerve atrophy. The causative gene, WFS1, encodes for Wolframin, a master regulator of several cellular responses, whose mutations associate with clinical variability. Indeed, nonsense/frameshift variants correlate with more severe symptoms than missense/in-frame ones. As achieving a genotype-phenotype correlation is crucial to deal with disease outcome, works investigating the impact of transcriptional and translational landscapes stemming from such mutations are needed. Therefore, we sought to elucidate the molecular determinants behind the pathophysiological alterations in a WS1 patient carrying compound heterozygous mutations in WFS1 gene: c.316-1G>A, affecting the acceptor splice site (ASS) upstream exon 4, and c.757A>T, introducing a premature termination codon (PTC) in exon 7. MethodsBioinformatic analysis was carried out to infer the alternative splicing events occurring after disruption of ASS, followed by RNAseq and PCR to validate the transcriptional landscape. Patient-derived induced Pluripotent Stem Cells (iPSCs) were used as an in vitro model of WS1 and to investigate the WFS1 alternative splicing isoforms into pancreatic {beta} cells. CRISPR/Cas9 technology was employed to correct ASS mutation and generate a syngeneic control for the ER-stress induction and immunotoxicity assays. ResultsWe showed that patient-derived iPSCs retained the ability to differentiate into pancreatic {beta} cells. We demonstrated that the allele carrying the ASS mutation c.316-1G>A originates two PTC-containing alternative splicing transcripts (c.316del and c.316-460del), and two ORF-conserving mRNAs (c.271-513del and c.316-456del) leading to N-terminally truncated polypeptides. By retaining the C-terminal domain, these isoforms sustained the endoplasmic reticulum (ER)-stress response in {beta} cells. Otherwise, PTC-carrying transcripts were regulated by the nonsense mediated decay (NMD) in basal conditions. Exposure to cell stress inducers and pro-inflammatory cytokines affected the NMD-related gene SMG7 expression levels (>2 fold decrease; p<0.001) without eliciting a robust unfolded protein response in WFS1 {beta} cells, thus resulting in a dramatic accumulation of the PTC-containing isoforms c.316del (>100-fold increase over basal; p<0.001) and c.316-460del (>20-fold increase over basal; p<0.001) and predisposing affected {beta} cells to undergo apoptosis. Cas9-mediated recovery of ASS retrieved the canonical transcriptional landscape, rescuing the normal phenotype in patient-derived {beta} cells. Conclusions/interpretationThis study represents a new model to study Wolframin, highlighting how each single mutation of WFS1 gene can determine dramatically different functional outcomes. Our data point to increased vulnerability of WFS1 {beta} cells to stress and inflammation, and we postulate that this is triggered by escaping NMD and accumulation of mutated transcripts and truncated proteins. These findings pave the way for further studies on the molecular basis of genotype-phenotype relationship in WS1, to uncover the key determinants that might be targeted to ameliorate the clinical outcome of patients affected by this rare disease.

genetics↗

bioRxiv2021

Follicular helper T cell signature of replicative exhaustion, apoptosis and senescence in common variable immunodeficiency

BackgroundCommon variable immunodeficiency (CVID) is the most frequent primary antibody deficiency. A significant number of CVID patients are affected by various manifestations of immune dysregulation such as autoimmunity. Follicular T cells cells are thought to support the development of CVID by providing inappropriate signals to B cells during the germinal center (GC) response. ObjectivesWe determined the possible role of follicular helper (Tfh) and follicular regulatory T (Tfr) cells in patients with CVID by phenotypic, molecular, and functional studies. MethodsWe analyzed the frequency, phenotype, transcriptome, and function of circulating Tfh cells in the peripheral blood of 27 CVID patients (11 pediatric and 16 adult) displaying autoimmunity as additional phenotype and compared them to 106 (39 pediatric and 67 adult) age-matched healthy controls. We applied Whole Exome Sequencing (WES) and Sanger sequencing to identify mutations that could account for the development of CVID and associate with Tfh alterations. ResultsA group of CVID patients (n=9) showed super-physiological frequency of Tfh1 cells and a prominent expression of PD-1 and ICOS, as well as a Tfh RNA signature consistent with highly active, but exhausted and apoptotic cells. Plasmatic CXCL13 levels were elevated in these patients and positively correlated with Tfh1 cell frequency, PD-1 levels, and an elevated frequency of CD21loCD38lo autoreactive B cells. Monoallelic variants in RTEL1, a telomere length- and DNA repair-related gene, were ideintified in four patients belonging to this group. Lymphocytes with highly shortened telomeres, and a Tfh signature enriched in genes involved in telomere elongation and response to DNA damage were seen. Histopathological analysis of the spleen in one patient showed reduced amount and size of the GC that, unexpectedly, contained an increased number of Tfh cells. ConclusionThese data point toward a novel pathogenetic mechanism in a group of patients with CVID, whereby alterations in DNA repair and telomere elongation might be involved in GC B cells, and acquisition of a Th1, highly activated but exhausted and apoptotic phenotype by Tfh cells.

immunology↗