Biology subjects

Spagnoli, F. M.

Publications and source records attributed to Spagnoli, F. M..

10 recordsLinked to original sources

bioRxiv2025

Mesodermal-niche interactions direct specification and differentiation of pancreatic islet cells in human multilineage organoids

Tissues develop and function within a highly complex microenvironment, where diverse cell types interact in tightly regulated spatial and temporal patterns1,2. Through distinct, relay-like waves of activity, these cells collectively shape tissue formation, ensuring that each component emerges in the right place at the right time1,2. Such coordinated cues establish the structural and biochemical framework that drives cell differentiation and tissue organization. Accurately modelling this process in humans requires the development of complex multicellular systems. Here, we pair spatial transcriptomics of the human foetal pancreas with an induced pluripotent stem cell (iPSC)-based multilineage model that faithfully recapitulate the complex, hierarchical processes underlying human pancreatic islet formation. We show that iPSC-derived pancreatic mesodermal lineages direct endocrine commitment from pancreatic progenitors, by suppressing off-target fates and orchestrating niche-mediated spatio-temporal cues that promote beta-cell differentiation. Our results identify a vascular-rich niche, featuring pancreatic pericytes, which is associated with a neural repulsion program and may contribute to shaping the islet microenvironment. We benchmark our in vitro multilineage organoid system against spatial transcriptomics of the human foetal pancreas. Together, these findings identify the key cellular actors and contact-dependent mechanisms that build the human endocrine pancreas, providing a critical model for studying human islet development and disease.

developmental biology↗

bioRxiv2025

Spatially organized cellular communities shape functional tissue architecture in the pancreas

Organ function depends on the precise spatial organization of cells across multiple scales, from individual cells to cellular communities that form specialized local niches and, ultimately, complex higher-order structures. While the identities of individual cell are increasingly well-defined, our understanding of how these diverse cell types are spatially distributed and communicate remains incomplete. In this study, we combine single-cell and spatial transcriptomic analyses to map pancreatic cell populations across space and time, from embryonic development to adult homeostasis in mice. Using these comprehensive maps, we systematically resolve spatial heterogeneity among pancreatic cell types and uncover basic tissue niches, which emerge as epithelial-mesenchymal units. We further characterize these niches functionally in both mouse and human models. We demonstrate that the mesenchymal lineage initially diversifies into various subtypes with specialized supportive roles during embryonic development. However, this complexity gradually diminishes over time, ultimately converging into a limited number of fibroblast sub-types in adult tissue. Our findings shed light on how different progenitor lineages co-develop and organize into structured communities that establish a mature, functional pancreas. This foundational framework could inform strategies for in vitro organogenesis and tissue-engineering in the context of pancreatic diseases.

developmental biology↗

bioRxiv2024

ΔNP63 defines an exocrine-committed multipotent progenitor subset in the murine pancreas

Cellular plasticity underpins heterogeneity in embryogenic progenitor cells and cancer cells. The transcription factor deltaNp63 ({Delta}Np63) has been implicated in regulating cellular plasticity in several epithelial tissues. Despite a recently established role in steering plasticity of pancreatic cancer, {Delta}Np63 remains unstudied in pancreatic development. Using murine single-cell sequencing data and RNA and protein in situ stainings, we assessed the spatio-temporal expression of Trp63 and {Delta}NP63 in the embryonic pancreas. {Delta}NP63 demonstrates a transient and spatially restricted expression in the multipotent pancreatic progenitor (MPP) compartment delineating pro-exocrine progenitor cells. Lineage tracing of TP63+ cells marks a subset of MPPs and descendant exocrine acinar and centro-acinar/terminal duct cells. Lack of {Delta}NP63 in knock-out mice leads to hypotrophic exocrine acini with reduced levels of differentiation markers. In summary, {Delta}Np63 confers heterogeneity within the MPP compartment, supporting exocrine cell development. These new insights in developmental plasticity have potential implications for pancreatic regeneration and cancer.

developmental biology↗

bioRxiv2024

Generation of human iPSC-derived pancreatic organoids to study pancreas development and disease

The pancreas has vital endocrine and exocrine functions that can be affected by life-threatening diseases such as diabetes and pancreatic cancer. While animal models have been essential for understanding pancreatic development and disease, they are limited by their low-throughput and major species-specific molecular and physiological differences. Generating 3D in vitro models, such as organoids, that are physiologically relevant is thus primordial for investigating pancreatic development and disease in the human context. However, producing human stem cell-derived pancreatic organoids with proper branched architecture and correct patterning of cell domains has remained challenging. Here, we successfully developed a protocol that efficiently and reproducibly generates such organoids from human induced pluripotent stem cells (hiPSCs) by optimising organoid culture format and media. Our differentiation protocol promotes acinar cell differentiation and generates organoids with branches patterned into central trunk and peripheral tip domains, without relying on animal-derived matrices for organoid culture. This platform opens the door to high-throughput investigations of human pancreas development in a system recapitulating the most important aspects of pancreatic tissue architecture. Lastly, we anticipate that this system will contribute to the partial replacement of animal models used to investigate diseases such as pancreatic cancer.

developmental biology↗

bioRxiv2023

Heterozygous missense variant in GLI2 impairs human endocrine pancreas development

Missense variants are the most common type of coding genetic variants. Their functional assessment is fundamental for defining any implication in human diseases and may also uncover genes that are essential for human organ development. Here, we applied CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant in GLI2 identified in a paediatric hyperglycaemic patient and family members. GLI2 is a primary mediator of the Hedgehog pathway, which regulates pancreatic {beta}-cell development in mice. However, neither mutations in GLI2 nor Hedgehog dysregulation have been reported as cause or predisposition to diabetes. A set of isogenic iPSC lines harbouring the missense variant were studied for their ability to differentiate into pancreatic {beta}-like cells. Interestingly, iPSCs carrying the missense variant showed altered GLI2 transcriptional activity and impaired endocrine differentiation. Collectively, our findings underscore an essential role for GLI2 in human endocrine development and identify a novel variant that predisposes to diabetes.

developmental biology↗

bioRxiv2023

Acinar-ductal cell rearrangement drives pancreas branching morphogenesis in an IGF/PI3K-dependent manner

During organ formation, progenitor cells need to acquire the diversity of cell identities found in the organ as well as organize themselves into distinct structural units. How these processes are coordinated, and how tissue architecture(s) are preserved despite the dramatic cell rearrangements occurring in developing organs remain unclear. Here, we identified cellular rearrangements between acinar and ductal progenitors as a mechanism to drive branching morphogenesis in the pancreas while preserving the integrity of the acinar-ductal functional unit. Using ex vivo and in vivo mouse models, we found that pancreatic ductal cells form clefts by protruding and pulling on the acinar basement membrane, which lead to acini splitting. Newly formed acini remain connected to bifurcated branches generated by ductal cell rearrangement. IGF/PI3K pathway regulates this process by controlling ductal cell fluidity. If components of the pathway are genetically or chemically dysregulated, ductal cell fluidity prevents branching and affects pancreatic cell fates. Hence, our results explain how acinar multiplication and branch bifurcation are synchronized during pancreas organogenesis.

developmental biology↗

bioRxiv2022

Improving stem cell-derived pancreatic islets using single-cell multiome-inferred regulomes

Pancreatic islet cells derived from human pluripotent stem cells hold great promise for modeling and treating diabetes. Differences between stem cell-derived and primary islets remain, but molecular insights to inform improvements are limited. Here, we acquire single-cell transcriptomes and accessible chromatin profiles during in vitro islet differentiation and pancreas from childhood and adult donors for comparison. We delineate major cell types, define their regulomes, and describe spatiotemporal gene regulatory relationships between transcription factors. CDX2 emerged as a regulator of enterochromaffin-like cells, which we show resemble a transient, previously unrecognized, CDX2+ pre-{beta}-cell population in fetal pancreas, arguing against a proposed non-pancreatic origin. Furthermore, we observe insufficient activation of signal-dependent transcriptional programs during in vitro {beta}-cell maturation and identify sex hormones as drivers of {beta}-cell proliferation in childhood. Altogether, our analysis provides a comprehensive understanding of cell fate acquisition in stem cell-derived islets and a framework for manipulating cell identities and maturity.

cell biology↗

bioRxiv2022

Identification and implication of tissue-enriched ligands in epithelial-endothelial crosstalk during pancreas development

Development of the pancreas is driven by an intrinsic program coordinated with signals from other cell types in the epithelial environment. These intercellular communications have been so far challenging to study because of the low concentration, localized production and diversity of the signals released. Here, we combined scRNAseq data with a computational interactomic approach to identify signals involved in the reciprocal interactions between the various cell types of the developing pancreas. This in silico approach yielded 40,607 potential ligand-target interactions between the different main pancreatic cell types. Among this vast network of interactions, we focused on three ligands potentially involved in communications between epithelial and endothelial cells. Bmp7 and Wnt7b, expressed by pancreatic epithelial cells and predicted to target endothelial cells, and Sema6d, involved in the reverse interaction. In situ hybridization confirmed the localized expression of Bmp7 in the pancreatic epithelial tip cells and of Wnt7b in the trunk cells. On the contrary, Sema6d was enriched in endothelial cells. Functional experiments on ex vivo cultured pancreatic explants indicated that tip cell-produced Bmp7 restrained development of endothelial cells. This work identified ligands with a restricted tissular and cellular distribution and highlighted the role of Bmp7 in the intercellular communications shaping vessel development during pancreas organogenesis.

developmental biology↗

bioRxiv2021

Development of a 3D atlas of the embryonic pancreas for topological and quantitative analysis of heterologous cell interactions

Generating comprehensive image maps, while preserving spatial 3D context, is essential to quantitatively assess and locate specific cellular features and cell-cell interactions during organ development. Despite the recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on 2D histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in 3D and map tissue interactions at key development time points in the mouse embryo. We used transgenic mouse models and antibodies to visualize the three main cellular components within the developing pancreas, including epithelial, mesenchymal and endothelial cell populations. We demonstrated the utility of the approach by providing volumetric data, 3D distribution of distinct progenitor populations and quantification of relative cellular abundance within the tissue. Lastly, our image data were combined in an open source online repository (referred to as Pancreas Embryonic Cell Atlas). This image dataset will serve the scientific community by enabling further investigation on pancreas organogenesis but also for devising strategies for the in vitro generation of transplantable pancreatic tissue for regenerative therapies.

developmental biology↗

bioRxiv2020

Quantitative lineage analysis identifies a long-term progenitor niche for the hepato-pancreato-biliary organ system

Single cell-based studies have revealed tremendous cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degree of plasticity during organogenesis1-5. The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including liver, pancreas, gallbladder, and extra-hepatic bile ducts6, 7. Experimental manipulation of various developmental signals in the mouse embryo underscored an important cellular plasticity in this embryonic territory6, 8. This is also reflected in the existence of human genetic syndromes as well as congenital or environmentally-caused human malformations featuring multiorgan phenotypes in liver, pancreas and gallbladder6, 8. Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary, and pancreatic structures are not yet established. Here, we combine computational modelling approaches with genetic lineage tracing to assess the tissue dynamics accompanying the ontogeny of the hepato-pancreato-biliary organ system. We show that a long-term multipotent progenitor domain persists at the border between liver and pancreas, even after pancreatic fate is specified, contributing to the formation of several organ derivatives, including the liver. Moreover, using single-cell RNA sequencing we define a specialized niche that possibly supports such long-term cell fate plasticity.

developmental biology↗