Biology subjects

Iglesias, M.

Publications and source records attributed to Iglesias, M..

18 recordsLinked to original sources

bioRxiv2026

Human SHED-derived extracellular cues activate a specialized neuroprotective and regenerative program in developing retinal ganglion cells

Axonal degeneration and insufficient neuronal survival remain major barriers to central nervous system repair. Stem cells from human exfoliated deciduous teeth (SHED) represent an accessible, developmentally immature, neural crest-derived mesenchymal stem cell population with potential relevance for neuroregenerative medicine. Here, we show that SHED display enhanced proliferative stability, preserved mesenchymal identity, and more sustained expansion capacity than adult dental pulp stem cells, supporting their suitability for scalable regenerative applications. Using embryonic chick retinal explants at neurogenic and post-neurogenic stages, we demonstrate that SHED robustly promote retinal ganglion cell axonogenesis, axonal regeneration, and neuronal survival. At embryonic day 5, SHED enhanced axonal outgrowth in both newly generated EdU/TUJ1 neurons and pre-existing EdU-/TUJ1 retinal ganglion cells. At embryonic day 13, when retinal neurons are post-mitotic and intrinsically less regenerative, SHED still significantly increased regenerative axonal extension and reduced developmental cell death. To investigate the molecular mechanisms underlying the neuroprotective and axogenic effects of SHED, proteomic profiling of SHED-retina co-culture secretomes was performed, revealing a highly enriched extracellular environment containing matrix-associated and neurodevelopmental proteins, including thrombospondin-1 (THBS1), galectin1 and 3, and multiple proteins associated with IGF2 pathway. Proteomic analysis of the SHED secretome, together with prior evidence implicating thrombospondin signaling in neuronal development and synaptogenesis, identified THBS1 as a strong candidate mediator of SHED-induced effects in chick retinal co-culture systems. Neutralization of THBS1, particularly in combination with gabapentin-mediated blockade of 2{delta}-1-dependent thrombospondin signaling, markedly reduced SHED-induced axonal growth and induced neuritic swellings consistent with impaired axonal integrity. In contrast, inhibition of THBS1 signaling did not significantly abolish the neuroprotective effect of SHED on neuronal survival, suggesting that distinct paracrine mechanisms independently regulate axonal regeneration and cell survival. Together, these findings demonstrate that SHED-derived combined secreted factors promote neuronal survival and axonal regeneration through partially divergent extracellular matrix-associated developmental pathways, positioning SHED and their secretome as promising candidates for cell-based and cell-free neuroregenerative strategies.

cell biology↗

bioRxiv2026

GATA4 loss promotes mutant Kras-driven pancreatic ductal adenocarcinoma in the absence of canonical precursor lesions

ABSTRACTGATA6 and GATA4 play key roles in pancreatic development and are essential to maintain the classical transcriptional program in pancreatic ductal adenocarcinoma (PDAC). Using genetic mouse models we show that, in contrast to GATA6, GATA4 is dispensable for the maintenance of acinar homeostasis in the adult pancreas. Deletion of Gata4 in mice expressing mutant Kras in the embryonic pancreas (KG4C) leads to PDAC development in the absence of tissue remodeling, pancreatic intraepithelial neoplasia (PanIN), or other canonical precursor lesions present in Gata4-proficient (KC) mice. Similar observations were made when Gata4 was selectively inactivated in adult, Kras-mutant, acinar cells. We identify Pale Acinar Lesions (PALes) as a previously unrecognized pancreatic lesion, distinct from acino-ductal metaplasia (ADM) and PanINs, present in KC and KG4C mice but not in wild type mice. PALes display weak expression of acinar and ductal markers and lack mucins; they have lower proliferation rates than PanINs. RNA-seq and ChIP-seq reveal that GATA4 and GATA6 partially share genomic binding sites and transcriptomic effects, but they exert opposing influences on mutant Kras-induced, haematopoietic cell-dependent, transcriptional inflammatory programs. Adenoviral-mediated pancreatic expression of IL17 restored the formation of ductal lesions in KG4C mice but failed to rescue PanIN development. Our data indicate that GATA4 functions through the coordinated action of multiple inflammatory factors that are required for ADM/PanIN formation but are dispensable for PDAC development. Collectively, these findings challenge current paradigms of PDAC initiation and progression.

cancer biology↗

bioRxiv2026

Evolutionary landscapes of zygotic genome activation across animals

During early animal embryogenesis, gene expression control shifts from maternally deposited products to newly transcribed RNA through zygotic genome activation (ZGA). Although essential and universal, ZGA remains poorly understood outside a few model species. Here, we generated a transcriptomic atlas of early embryogenesis across 61 animal species from 13 phyla and used a unified computational framework to infer ZGA timing systematically. We find extensive variation in ZGA onset across animals, but show that the ratio between genome size and egg cytoplasmic volume, a proxy for the nuclear-to-cytoplasmic ratio, robustly predicts when genome activation begins. We also reveal that zygotic genes also differ from maternal transcripts in genomic architecture, function and evolutionary conservation, suggesting that ZGA follows a conserved molecular logic despite flexible transcriptomic outputs.

evolutionary biology↗

bioRxiv2026

Life-stage-specific specialities in the cell atlases of the Clytia hemisphaerica planula and medusa

Jellyfish have complex life-cycles, but there has been limited exploration of how this is achieved at the cellular level. We used single-cell transcriptomics to assemble a cell atlas for the planula larva of Clytia hemisphaerica, and compared it to an updated cell atlas for the medusa (jellyfish) stage. The cells of the planula fell into the same broad categories as for the medusa: ectoderm, gastroderm, interstitial cells (i-cells), nematocytes (stinging cells), neurons and secretory cells. Although the planula cells generally showed less diversity than medusae within each category, cells with specialized features unique to their stage could be distinguished by their transcriptional profiles as well as by ultrastructure. Some planula-specific types were identified: aboral secretory cells involved in settlement, and a cell type attributed a role in immunity or post-metamorphic theca production. Distinct transcriptome profiles within different regions of the ciliated planula ectoderm reflected different post-metamorphosis fates of domains along the oral-aboral axis. Inspection of the cell clusters showing significant similarity of marker genes between planula and medusa, and inference of similarity using a statistical model of marker gene presence/absence, revealed correspondences between families of cells from planula and medusa rather than precise cell identities.

evolutionary biology↗

bioRxiv2025

Decoding cnidarian cell type gene regulation

Animal cell types are defined by differential access to genomic information, a process orchestrated by the combinatorial activity of transcription factors that bind to cis-regulatory elements (CREs) to control gene expression. However, the regulatory logic and specific gene networks that define cell identities remain poorly resolved across the animal tree of life. As early-branching metazoans, cnidarians can offer insights into the early evolution of cell type-specific genome regulation. Here, we profiled chromatin accessibility in 60,000 cells from whole adults and gastrula-stage embryos of the sea anemone Nematostella vectensis. We identified 112,728 CREs and quantified their activity across cell types, revealing pervasive combinatorial enhancer usage and distinct promoter architectures. To decode the underlying regulatory grammar, we trained sequence-based models predicting CRE accessibility and used these models to infer ontogenetic relationships among cell types. By integrating sequence motifs, transcription factor expression, and CRE accessibility, we systematically reconstructed the gene regulatory networks that define cnidarian cell types. Our results reveal the regulatory complexity underlying cell differentiation in a morphologically simple animal and highlight conserved principles in animal gene regulation. This work provides a foundation for comparative regulatory genomics to understand the evolutionary emergence of animal cell type diversity.

genomics↗

bioRxiv2024

T cell responsiveness to IL-10 defines the immunomodulatory effect of costimulation blockade via anti-CD154 and impacts transplant survival

Costimulation blockade (CoB)-based immunotherapy is a promising alternative to immunosuppression for transplant recipients; however, the current limited understanding of the factors that impact its efficacy restrains its clinical applicability. In this context, pro- and anti-inflammatory cytokines are being recognized as having an impact on T cell activation beyond effector differentiation. This study aims at elucidating the impact of direct IL-10 signaling in T cells on CoB outcomes. We used a full-mismatch skin transplantation model where recipients had a T cell-restricted expression of a dominant negative IL-10 receptor (10R-DN), alongside anti-CD154 as CoB therapy. Unlike wild-type recipients, 10R-DN mice failed to benefit from CoB. This accelerated graft rejection correlated with increased accumulation of T cells producing TNF-, IFN-{gamma}, and IL-17. In vitro experiments indicated that while lack of IL-10 signaling did not change the ability of anti-CD154 to modulate alloreactive T cell proliferation, the absence of this pathway heightened TH1 effector cell differentiation. Furthermore, deficiency of IL-10 signaling in T cells impaired Treg induction, a hallmark of anti-CD154 therapy. Overall, these findings unveil an important and novel role of IL-10 signaling in T cells that defines the success of CoB therapies and identifies a target pathway for obtaining robust immunoregulation.

immunology↗

bioRxiv2023

Human Placenta-Derived Mesenchymal Stem Cells Stimulate Neuronal Regeneration By Promoting Axon Growth And Restoring Neuronal Activity

Cell therapy is a cutting-edge medical approach that involves the use of cells to treat various diseases and conditions. It harnesses the remarkable regenerative and reparative abilities of cells to restore or replace damaged tissues and promote healing. In the last decades, mesenchymal stem cells (MSCs) have become the cornerstone of cellular therapy due to their unique characteristics. Specifically human placenta-derived mesenchymal stem cells (hPMSCs) are highlighted for their unique features, including ease to isolate, non-invasive techniques for large scale cell production, significant immunomodulatory capacity, and a high ability to migrate to injuries. Researchers are exploring innovative techniques to overcome the low regenerative capacity of Central Nervous System (CNS) neurons, with one promising avenue being the development of tailored mesenchymal stem cell therapies capable of promoting neural repair and recovery. In this context, we have evaluated hPMSCs as candidate for CNS lesion regeneration using a skillful co-culture model system. Indeed, we have demonstrated the hPMSCs ability to stimulate damaged rat-retina neurons regeneration by promoting axon growth and restoring neuronal activity both under normoxia and hypoxia conditions. With our model we have obtained neuronal regeneration values of 10-12% and axonal length per neuron rates of 19.99{+/-}0.77, m/neuron. To assess whether the regenerative capabilities of hPMSCs are contact-dependent effects or it is mediated through paracrine mechanisms, we carried out transwell co-culture and conditioned medium experiments confirming the role of secreted factors in axonal regeneration. It was found that hPMSCs produce brain derived, nerve growth factors (BDNF, NGF) and Neurotrophin-3, involved in the process of neuronal regeneration and restoration of their physiological activity of neurons. The capability to access axonal physiology is crucial for studying information processing among neurons in healthy and diseased states. We confirm the success of our treatment using the patch clamp technique to study ionic currents in individual isolated living cells, confirming that in our model the regenerated neurons are electrophysiologically active.

cell biology↗

bioRxiv2023

A Functional Exodermal Suberin is Key for Plant Nutrition and Growth in Potato

Angiosperm roots, except in Arabidopsis, have both endodermis and exodermis, which regulate radial water and solute movement through lignin and suberin deposition. While endodermal suberin in Arabidopsis acts as a barrier to water and solute uptake and backflow, its implications in other angiosperms with both layers and the role of exodermal suberin remain unclear. We examined potato roots (Solanum tuberosum) and found that exodermis lacks the typical Casparian strip but forms an outer lignin cap, and quickly suberizes near the root tip. In contrast, a few endodermal cells, with Casparian strip, start suberizing much later. The continuous early exodermal suberization covering the root underlines its potential role in mineral nutrient radial movement. To demonstrate it, we used plants downregulating the suberin biosynthetic gene CYP86A33, which had the root suberin reduced in a 61%. Phenotypic analyses of the suberin-deficient mutant showed altered mineral nutrient concentration, slightly reduced water content and compromised growth. Micro-PIXE analyses identified the distribution of elements within the roots and highlighted anatomical compartments defined by apoplastic barriers. These findings advance our understanding of nutrient radial transport, demonstrate exodermal suberin as a bidirectional and selective barrier to element movement, and underscore its importance in nutrient homeostasis and plant growth.

plant biology↗

bioRxiv2023

IKK1 kinase coordinates BRD4 and JAK/STAT signaling to subvert DNA damage-based anticancer therapy

Activation of the IKK kinase complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-{kappa}B has remained elusive. Analysis of IKK-dependent substrates after UV-treatment revealed that BRD4 phosphorylation by IKK is required for chromatin-binding dynamics upon damage. Moreover, IKK induces the NF-{kappa}B-dependent transcription of LIF leading to STAT3 activation, association of BRD4 to STAT3 and recruitment to specific target genes. IKK abrogation results in defective BRD4 and STAT3 function leading to irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK activity or BRD4 and the JAK/STAT pathway enhanced the therapeutic potential of 5-FU plus irinotecan in CRC cells, and is curative in a chemotherapy-resistant CRC xenograft model. Coordinated expression of LIF and IKK is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK, BRD4 and JAK/STAT signaling with clinical relevance.

cancer biology↗

bioRxiv2022

Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection

Low dose human interleukin-2 (hIL-2) treatment is used clinically to treat autoimmune disorders due to the cytokines preferential expansion of immunosuppressive regulatory T cells (TRegs). However, high toxicity, short serum half-life, and off-target immune cell activation limit the clinical potential of IL-2 treatment. Recent work showed that complexes comprising hIL-2 and the anti-hIL-2 antibody F5111 overcome these limitations by preferentially stimulating TRegs over immune effector cells. Although promising, therapeutic translation of this approach is complicated by the need to optimize dosing ratios and by the instability of the cytokine/antibody complex. We leveraged structural insights to engineer a single-chain hIL-2/F5111 antibody fusion protein, termed F5111 immunocytokine (IC), that potently and selectively activates and expands TRegs. F5111 IC conferred protection in mouse models of colitis and checkpoint inhibitor-induced diabetes mellitus. These results provide a roadmap for IC design and establish a TReg-biased immunotherapy that could be clinically translated for autoimmune disease treatment.

bioengineering↗

bioRxiv2021

Reversing chemorefraction in colorectal cancer cells by controlling mucin secretion

15% of colorectal cancers (CRC) cells exhibit a mucin hypersecretory phenotype, which is suggested to provide resistance to immune surveillance and chemotherapy. We now formally show that colorectal cancer cells build a barrier to chemotherapeutics by increasing mucins secretion. We show that low levels of KChIP3, a negative regulator of mucin secretion (Cantero-Recasens et al., 2018), is a risk factor for CRC patients relapse in subset of untreated tumours. Our results also reveal that cells depleted of KChIP3 are four times more resistant (measured as cell viability and DNA damage) to chemotherapeutics 5-Fluorouracil plus Irinotecan (5-FU+iri.) compared to control cells, whereas KChIP3 overexpressing cells are 10 times more sensitive to killing by chemotherapeutics. Similar increase in tumour cell death is observed upon chemical inhibition of mucin secretion by the sodium/calcium exchanger (NCX) blockers (Mitrovic et al., 2013). Finally, sensitivity of CRC patient-derived organoids to 5-FU+iri increases 40-fold upon mucin secretion inhibition. Reducing mucin secretion thus provides a means to control chemoresistance of mucinous colorectal cancer cells and other mucinous tumours.

cell biology↗

bioRxiv2021

Chemotherapy induces a YAP1-dependent fetal conversion to human Colorectal Cancer cells that is predictive of poor patient outcome

Current therapy against colorectal cancer is based on DNA-damaging agents that eradicate highly proliferative malignant cells. Whether sublethal chemotherapy affects tumor cell behavior and impacts on patient outcome is primarily unstudied. We now show that sublethal chemotherapy imposes a quiescent-like state to p53 wildtype human colorectal cancer (CRC) cells that is linked to the acquisition of a fetal phenotype downstream of YAP1, similar to that observed after intestinal damage. CRC cells displaying this fetal phenotype exhibit tumor- initiating activity comparable to untreated cells but superior metastatic capacity. Notably, nuclear YAP1 accumulation, or detection of the fetal signature in tumors predict poor prognosis in CRC patients carrying p53 wildtype tumors. Collectively, our results uncover a potential adverse response of tumor cells to suboptimal chemotherapy, and identify nuclear YAP1 and fetal conversion of colorectal tumors as biomarkers for prognosis and therapy prescription. Statement of significanceChemotherapy induces a quiescent-like phenotype to colorectal cancer cells that is linked to the acquisition of a YAP1-dependent fetal signature. Notably, this signature is predictive of patient outcome in different cohorts of human colorectal cancer.

cancer biology↗

bioRxiv2020

A short course of Tofacitinib sustains the immunoregulatory effect of CTLA4-Ig in presence of inflammatory cytokines and promotes long-term survival of murine cardiac allografts

Costimulation blockade-based regimens are a promising strategy for management of transplant recipients. However, maintenance immunosuppression via CTLA4-Ig monotherapy is characterized by high frequency of rejection episodes. Recent evidence suggests that inflammatory cytokines contribute to alloreactive T cell activation in a CD28-independent manner, a reasonable contributor to the limited efficacy of CTLA4-Ig. In this study, we investigated the possible synergism of a combined short-term inhibition of cytokine signaling and CD28 engagement on the modulation of rejection. Our results demonstrate that the JAK/STAT inhibitor Tofacitinib restored the immunomodulatory effect of CTLA4-Ig on mouse alloreactive T cells in presence of inflammatory cytokines. Tofacitinib exposure conferred dendritic cells with a tolerogenic phenotype reducing their cytokine secretion and costimulatory molecules expression. JAK inhibition also directly affected T cell activation. In vivo, the combination of CTLA4-Ig and Tofacitinib induced long-term survival of heart allografts and, importantly, it was equally effective when using grafts subjected to prolonged ischemia. Transplant survival correlated with a reduction in effector T cells and intragraft accumulation of regulatory T cells. Collectively, our studies demonstrate a powerful synergism between CTLA4-Ig and Tofacitinib and suggest their combined use is a promising strategy for improved management of transplanted patients.

immunology↗

bioRxiv2020

ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics

Single-cell sequencing technologies are revolutionizing biology, but are limited by the need to dissociate fresh samples that can only be fixed at later stages. We present ACME (ACetic-MEthanol) dissociation, a cell dissociation approach that fixes cells as they are being dissociated. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, can be sorted by Fluorescence-Activated Cell Sorting (FACS) and are permeable, enabling combinatorial single-cell transcriptomic approaches. As a proof of principle, we have performed SPLiT-seq with ACME cells to obtain around ∼34K single cell transcriptomes from two planarian species and identified all previously described cell types in similar proportions. ACME is based on affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2020

A multilayered post-GWAS assessment on genetic susceptibility to pancreatic cancer

Pancreatic cancer (PC) is a complex disease in which both non-genetic and genetic factors interplay. To-date, 40 GWAS hits have been associated with PC risk in individuals of European descent, explaining 4.1% of the phenotypic variance. Here, we complemented a classical new PC GWAS (1D) with spatial autocorrelation analysis (2D) and Hi-C maps (3D) to gain additional insight into the inherited basis of PC. In-silico functional analysis of public genomic information allowed prioritization of potentially relevant candidate variants. We replicated 17/40 previous PC-GWAS hits and identified novel variants with potential biological functions. The spatial autocorrelation approach prioritized low MAF variants not detected by GWAS. These were further expanded via 3D interactions to 54 target regions with high functional relevance. This multi-step strategy, combined with an in-depth in-silico functional analysis, offers a comprehensive approach to advance the study of PC genetic susceptibility and could be applied to other diseases.

genetics↗

bioRxiv2019

Systematic Development Of Sandwich Immunoassays For The Plasma Secretome

The plasma proteome offers a clinically useful window into human health and disease. With recent progress made on the development of highly multiplexed immunoassays with high sample throughput, a remaining need is to establish a pipeline for validating the individual proteins that build such bio-signatures by using targeted assays. In order to streamline such efforts, we developed a workflow to build dual binder sandwich immunoassays (SIA) and chose to evaluate this on proteins predicted to be secreted form cells and tissues. Utilizing the multiplexing capacities of the bead array technology, we first screened ~ 1,800 unique antibody pairs against 209 protein targets and collected data from dilution series of recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies from the Human Protein Atlas, we obtained dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets. For 22 protein assays, the longitudinal, inter-individual and technical performance was determined in a set of plasma samples collected from 18 healthy subjects every third month over one year. Lastly, we compared 14 of these assays with SIAs composed of other binders, proximity extension assays and affinity-free targeted mass spectrometry. Our workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, while the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

biochemistry↗

bioRxiv2018

Type-I interferons inhibit interleukin-10 signaling and favor type 1 diabetes development in NOD mice

Destruction of insulin-producing {beta}-cells by autoreactive T lymphocytes leads to the development of type 1 diabetes. Type I interferons (TI-IFN) and interleukin-10 (IL-10) have been connected with the pathophysiology of this disease; however, their interplay in the modulation of diabetogenic T cells remains unknown. We have discovered that TI-IFN cause a selective inhibition of IL-10 signaling in effector and regulatory T cells, altering their responses. This correlates with diabetes development in NOD mice, where the inhibition is also spatially localized to T cells of pancreatic and mesenteric lymph nodes. IL-10 signaling inhibition is reversible and can be restored via blockade of TI-IFN/IFN-R interaction, paralleling with the resulting delay in diabetes onset and reduced severity. Overall, we propose a novel molecular link between TI-IFN and IL-10 signaling that helps better understand the complex dynamics of autoimmune diabetes development and reveals new strategies of intervention.\n\nAbbreviations

immunology↗

bioRxiv2017

Opponent assessment and conflict resolution through mutual motor coordination

Most animals fight by repeating complex stereotypic behaviors, yet the internal structure of these behaviors has rarely been dissected in detail. We characterized the internal structure of fighting behaviors by developing a machine learning pipeline that measures and classifies the behavior of individual unmarked animals on a sub-second timescale. This allowed us to quantify several previously hidden features of zebrafish fighting strategies. We found strong correlations between the velocity of the attacker and the defender indicating a dynamic matching of approach and avoidance efforts consistent with the cumulative assessment model of aggression. While velocity matching was ubiquitous, the spatial dynamics of attacks showed phase-specific differences. Contest phase attacks were characterized by a paradoxical side-ways attraction of the retreating animal towards the attacker suggesting that the defender combines avoidance maneuvers with display maneuvers. Post-resolution attacks lacked display-like features and the the defender was avoidance-focused. From the perspective of the winner, game theory modeling further suggested that highly energetically costly post resolution attacks occurred because the winner was trying to increase its relative dominance over the loser. Overall, the rich structure of zebrafish motor coordination during fighting indicates a greater complexity and layering of strategies than has previously been recognized.

animal behavior and cognition↗