Biology subjects

Costagliola, S.

Publications and source records attributed to Costagliola, S..

13 recordsLinked to original sources

bioRxiv2026

Microphysiological Flow Batteries For Dynamic EDC Screening Of mESC-derived Thyroid Organoids

Endocrine disrupting chemicals (EDCs) are ubiquitous environmental contaminants capable of dysregulating the production of thyroid hormones. Traditional thyroid toxicological assays rely on 2D cell cultures and animal models, both of which fail to accurately recapitulate human thyroid physiology and provide limited mechanistic insight into EDC toxicity. To overcome these limitations, we report a novel thyroid-on-chip platform integrating mouse embryonic stem cell-derived thyroid organoids with advanced organ-on-chip (OoC) technology and downstream multi-omics analysis. The platform leverages a reversibly-sealed microphysiological flow battery (MFB) to allow scale up of dynamic organoid culture and controlled chemical exposure while reducing operational complexity. Upon EDC exposure, transcriptomic and proteomic analysis revealed new molecular signatures of thyroid disruption across four different EDC classes, even at very low EDC concentrations (1nM), validating the capacity of this system to mechanistically dissect EDC-induced responses. This represents an integrated platform consists of an advanced physiologically relevant assay framework for next-generation endocrine toxicity testing, bridging the gap between in vitro screening and in vivo thyroid physiology.

bioengineering↗

bioRxiv2026

Microfluidic bioprinting of a physiologically relevant thyroid three-dimensional in vitro model

Endocrine disruptors (EDs) are an exogenous group of compounds associated with thyroid malfunctioning in the human body. Nonetheless, there are currently no adequate in vivo or in vitro models for the preclinical testing of these compounds since both animal and two-dimensional (2D) cell-based models are not able to mimic thyroid physiological conditions from both functional and three-dimensional (3D) organization perspective. Recently, bioprinting technologies emerged as an innovative tool in the field of regenerative medicine and advanced 3D in vitro models that allow the creation of 3D well-organized structures able to mirror physiologically relevant tissue and organ architectures. In this study, we evaluated microfluidic bioprinting as a biofabrication technology to develop a 3D in vitro model of the thyroid gland. We studied the fundamental parameters to obtain a fine control over the bioprinted fibres for different biomaterials. Then, we assessed the possibility to bioprint single thyroid cells, thyroid spheroids and finally mouse embryonic stem cell-derived thyroid follicles. The different cell types maintained high viability and metabolic activity. The bioprinted thyroid model showed high expression of different early and late functional markers and to be responsive to ED exposure. These bioprinted thyroid constructs could provide a new set of advanced 3D in vitro models to test potential EDs and possible adverse outcomes that may be associated with their administration or exposure.

bioengineering↗

bioRxiv2025

Long-term prevention of aneuploidy in human pluripotent stem cells by fine-tuning GSK3 activity

Human pluripotent stem cells (hPSCs) are critical cell sources to model human development and hold promise towards regenerative medicine. However, cultured hPSCs quickly acquire chromosome gains or losses (aneuploidy) due to high replicative stress and errors in chromosome segregation, which hampers their use for stem cell therapies. We have recently described that embryo patterning signals control chromosome segregation fidelity during early human lineage specification by modulating the response to replicative stress. Here, we demonstrate that fine-tuned activation of WNT signalling using off-the-shelf GSK3 inhibitors prevents chromosome missegregation and aneuploidy during long term passaging of hPSCs. Lineage-specification and sequencing analyses demonstrate that hPSCs culture with E8 supplemented with a low dosage of GSK3 inhibitors retain primed pluripotency and full potential to the differentiation into the 3 germ layers for over 30 passages. Strikingly, detailed mitotic studies revealed that long-term cultured hPSCs often display ultra-fine chromosome bridges during anaphase due to unreplicated DNA, which can result in hitherto uncharacterised copy-number-variations and other genomic aberrations. We show that culture with low dosage of the GSK3 inhibitor CHIR99021, but not direct attenuation of DNA replication stress using nucleosides, prevents mitotic DNA synthesis and the formation of ultra-fine bridges during long term hPSC passage. Taken together, we propose to enrich E8 culture media with 100 nM CHIR99021 to maintain euploidy in primed hPSCs, and to routinely map for genomic alterations, in addition to aneuploidy, for modelling and regenerative medicine studies.

cell biology↗

bioRxiv2024

Single nuclei and spatial transcriptomes suggest a stratification of papillary and anaplastic thyroid cancer cells

Sixty percent of papillary thyroid cancers (PTCs) are driven by BRAFV600E, a mutation associated with high inter- and intra-tumoral heterogeneity. PTCs may become highly aggressive anaplastic thyroid cancers (ATC). While single cell transcriptomics may resolve this heterogeneity, it is potentially confounded by technical effects whose correction may dampen inter-tumor variations. Here we profiled ATCs and BRAFV600E PTCs with single nuclei RNA-seq and spatial transcriptomics, and an experimental design disentangling biological and technical variations. It reveals that much transcriptional variation in cancer cells and several immune cell types is idiosyncratic, i.e. tumor-specific, a phenomenon obscured by batch integration in a number of single cell studies. Idiosyncrasies are associated in some cases with genomic aberrations and global tissue states like hypoxia. Beyond idiosyncrasies, differentiation markers SLC5A5 (NIS), TPO, TG and TSHR are lost in a sequence mirrored by their gain during human thyroid organoids maturation, suggesting a new classification of cancer cell states. PTC cells retain TSHR expression and show features of partial EMT with a massive expression of FN1, which promotes proliferation via an autocrine loop. In contrast, ATCs undergo full blown EMT, with expression of mesenchymal extracellular components and loss of TSHR. Finally, we show that the microenvironment of cancer cells is driven by inflammation. These findings may help future stratifications of BRAFV600E PTCs.

cancer biology↗

bioRxiv2023

The highly and perpetually upregulated thyroglobulin gene is a hallmark of functional thyrocytes

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e. for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a valuable model to study the maintenance of a high transcriptional upregulation. SUMMARY STATEMENTThe thyroglobulin gene is highly and permanently expressed in thyrocytes of all vertebrates, at any condition and round the clock, offering a unique model to study mechanisms of high upregulation maintenance and chromatin dynamics

cell biology↗

bioRxiv2023

Modeling Braf-induced thyroid cancer development and drug screening using pluripotent stem cell-derived organoids

Thyroid cancer is the most common endocrine malignancy and several genetic events have been described to promote the development of thyroid carcinogenesis. Besides the effects of specific mutations on thyroid cancer development, the molecular mechanisms controlling tumorigenesis, tumor behavior, and drug resistance are still largely unknown. Cancer organoids have been proposed as a powerful tool to study aspects related to tumor development and progression and appear promising to test individual responses to therapies. Here, using mESC-derived thyroid organoids, we developed a BrafV637E- inducible model able to recapitulate the features of papillary thyroid cancer in vitro. Overexpression of the murine BrafV637E mutation, equivalent to BrafV600E in humans, rapidly triggers to MAPK activation, cell dedifferentiation, and disruption of follicular organization. BrafV637E-expressing organoids show a transcriptomic signature for p53, focal adhesion, ECM-receptor interactions, EMT, and inflammatory signaling pathways. Finally, PTC-like thyroid organoids were used for drug screening assays. The combination of MAPK and PI3K inhibitors reversed BrafV637E oncogene-promoted cell dedifferentiation while restoring thyroid follicle organization and function in vitro. Our results demonstrate that pluripotent stem cells-derived thyroid cancer organoids can mimic tumor development and features while providing an efficient tool for testing novel targeted therapies.

cancer biology↗

bioRxiv2022

Foxe1 orchestrates thyroid and lung cell lineage divergence in mouse stem cell-derived organoids

Patterning of endoderm into lung and thyroid lineages depends upon a correct early expression of a homeobox domain-containing transcription factor, Nkx2-1. However, the gene networks distinguishing the differentiation of those lineages remain largely unknown. In the present work, by using mouse embryonic stem cell lines, single-cell RNA sequencing, and transcriptomic and chromatin accessibility profiling, we show that knockout of Foxe1 drastically impairs Nkx2-1+ cells differentiation and maturation into thyroid follicular-like cells. Concomitantly, a subset of Foxe1 null/Nkx2-1+ cells have a remarkable ability in vitro to undergo a lung epithelial differentiation program and form lung-like organoids harboring cells transcriptionally similar with mouse fetal airway and alveolar cell types. These results demonstrate, for the first time, lung lineage derivation at the expense of thyroid lineage, by a simple removal of a transcription factor, and provide insights into the intricated mechanisms of fate decisions of endodermal cell types. Highlights- Forward programming of mESCs with transient Nkx2-1 and Pax8 overexpression, followed by c-AMP treatment, leads to differentiation of functional thyroid follicles in vitro; - In absence of Foxe1, thyroid follicle-like structures, derived from mESCs, are scarce and non-functional; - Concomitantly, a subset of Nkx2-1-expressing cells generated from Foxe1KO mESCs spontaneously form lung organoids containing multiple differentiated lung cell types; - ATACseq analyses show higher chromatin remodeling in Nkx2-1-expressing cells in control compared to Foxe1KO cells, especially for genes involved in thyroid maturation and maintenance of the 3D structure of the follicle.

developmental biology↗

bioRxiv2021

Transplantable human thyroid organoids generated from embryonic stem cells to rescue hypothyroidism

The thyroid gland captures iodide in order to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to hypothyroidism, which is one of the most common disorder in humans and is not always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, functional human thyroid organoids could pave the way to explore new therapeutic approaches. Here we report the first transplantable thyroid organoids derived from human embryonic stem cells capable of restoring plasma thyroid hormone to athyreotic mice as a proof of concept for future therapeutic development.

developmental biology↗

bioRxiv2021

Single-cell trajectory inference guided enhancement of thyroidmaturation in vitro using TGF-beta inhibition

The thyroid gland regulates metabolism and growth via secretion of thyroid hormones by thyroid follicular cells (TFCs). Loss of TFCs, by cellular dysfunction, autoimmune destruction or surgical resection, underlies hypothyroidism. Recovery of thyroid hormone levels by transplantation of mature TFCs derived from stem cells in vitro holds great therapeutic promise. However, the utilization of in vitro derived tissue for regenerative medicine is restricted by the efficiency of differentiation protocols to generate mature organoids. Here, to improve the differentiation efficiency for thyroid organoids, we utilized single-cell RNA-Seq to chart the molecular steps undertaken by individual cells during the in vitro transformation of mouse embryonic stem cells to TFCs. Our single-cell atlas of mouse organoid systematically and comprehensively identifies, for the first time, the cell types generated during production of thyroid organoids. Using pseudotime analysis, we identify TGF-beta and planar-cell polarity (PCP) pathways as regulators of thyroid maturation in vitro. Using pharmacological manipulation of TGF-beta pathway, we improve the level of thyroid maturation, in particular the induction of Nis expression. This in turn, leads to an enhancement of iodide organification in vitro, suggesting functional improvement of the thyroid organoid. Our study highlights the potential of single-cell molecular characterization in understanding and improving thyroid maturation and paves the way for identification of therapeutic targets against thyroid disorders.

developmental biology↗

bioRxiv2020

Timed mesodermal FGF and BMP govern the multi-step thyroid specification

Thyroid tissue is the site for de novo synthesis of thyroid hormones which are essential for vertebrate development and growth. Defects in embryonic thyroid morphogenesis are a predominant cause for congenital thyroid diseases but the molecular pathomechanisms are incompletely understood. The first molecularly recognizable step of thyroid development is the specification of thyroid precursors at a defined position in the anterior foregut endoderm. While recent studies identified FGF and BMP pathways as critical signaling factors for thyroid specification, the interplay between extrinsic signaling cues and thyroid transcription factor expression remained elusive. Here, we used zebrafish embryos to decipher the dynamics of thyroid transcription factor induction in relation to FGF and BMP signaling activities in pharyngeal endoderm. We first identified a previously unrecognized endodermal thyroid progenitor cell population expressing Pax2a but not Nkx2.4b. This cell population is characterized by enhanced FGF signaling but initially lacks detectable BMP signaling. A subpopulation of Pax2a-expressing progenitors differentiates subsequently into thyroid lineage-committed precursor cells co-expressing Pax2a and Nkx2.4b. We next combined pharmacological approaches with genetic models permitting inhibition or ectopic overactivation of signaling pathways to timely manipulate FGF and BMP activities. These experiments support a model where FGF signaling primarily regulates Pax2a expression whereas BMP signaling has dual functions in regulation of both Pax2a and Nkx2.4b expression. Collectively, our data allow us to formulate a refined model of thyroid cell specification from foregut endoderm.

developmental biology↗

bioRxiv2020

Single-cell transcriptome analysis reveals cell-cell communication and thyrocyte diversity in the zebrafish thyroid gland

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover genetic heterogeneity in the thyrocyte population, and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells and fibroblasts. Further, the thyrocytes could be split into two sub-populations with unique transcriptional signature, including differential expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line, which demonstrated specific pax2a expression in the thyrocytes. However, a population of pax2a-low mature thyrocytes interspersed within individual follicles could be distinguished, corroborating heterogeneity within the thyrocyte population. Our results identify and validate transcriptional differences within the nominally homogenous thyrocyte population. One-line summarySingle-cell analysis uncovers latent heterogeneity in thyroid follicular cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/891630v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@f6df3org.highwire.dtl.DTLVardef@fb8c59org.highwire.dtl.DTLVardef@bb5be1org.highwire.dtl.DTLVardef@8d174e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

Enhanced canonical Wnt signaling during early zebrafish development perturbs the interaction of cardiac mesoderm and pharyngeal endoderm and causes thyroid specification defects

BackgroundCongenital hypothyroidism (CH) due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the far majority of cases. This situation reflects in part our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification. MethodsWe treated zebrafish embryos during different developmental periods with small molecule compounds known to modulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm and cardiovascular development using whole mount in situ hybridization and transgenic fluorescent reporter models. We used an antisense morpholino to create a zebrafish acardiac model. For thyroid rescue experiments, BMP pathway induction in zebrafish embryos was obtained by using heatshock inducible transgenic lines. ResultsInterestingly, combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of morpholino-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development. ConclusionCollectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.

developmental biology↗

bioRxiv2019

Small Molecule Screening in Zebrafish Embryos Identifies Signaling Pathways Regulating Early Thyroid Development

BackgroundDefects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification and subsequent morphogenetic processes in thyroid development. MethodsTo begin to address this problem in a systematic way, we used zebrafish embryos to perform a series of in vivo phenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm and cardiovascular development using whole mount in situ hybridization and transgenic fluorescent reporter models. ResultsSystematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding as well as hypoplastic, enlarged or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified BMP and FGF signaling as key factors for thyroid specification and early thyroid organogenesis, highlight the importance of low Wnt activities during early development for thyroid specification and implicate drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis. ConclusionsBy integrating the outcome of our screening efforts with previously available information from other model organisms including Xenopus, chicken and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases.

developmental biology↗