Francesca Maria Spagnoli

Aims: Type 1 diabetes results from autoimmune destruction of insulin‐producing pancreatic β‐cells. Although exogenous insulin remains the standard treatment, it does not restore physiological glucose regulation. Differentiation of human pluripotent stem cells into β‐cells offers a scalable alternative to cadaveric islet transplantation and has progressed rapidly over the past decade, resulting in nine clinical trials at various stages of completion. Despite this progress, durable graft function remains constrained by two major challenges: the absence of key islet microenvironment components, such as vasculature, extracellular matrix and supporting cell types and continued vulnerability to autoimmune and alloimmune rejection. Methods & Results: This review considers emerging technological approaches to overcome these barriers. Bioengineered niches, including multilineage organoids, functionalized hydrogels, decellularized matrices and bioprinting, aim to recreate the native islet microenvironment, while synthetic biology approaches, such as hypoimmune gene editing, CRISPR‐based transcriptional control and stimulus‐responsive survival circuits, seek to enhance grafts' resilience. Computational and single‐cell approaches, including gene regulatory network analysis and digital twin frameworks, are beginning to provide predictive tools for benchmarking differentiation quality and forecasting graft outcomes. Conclusion: Together, these complementary strategies, potentially tailored to individual patients, represent a realistic path towards safer, more effective and durable β‐cell replacement therapies for type 1 diabetes.

Pancreatic development is a dynamic process shaped by coordinated interactions between surrounding non-pancreatic tissues and intrinsic transcription factors, integrated through gene regulatory networks. Advances in single-cell genomics and lineage-tracing technologies have substantially expanded our understanding of pancreatic biology, elucidating the ontogeny of pancreatic cells, their diversity and the intercellular interactions that govern them. In this Review, we examine the emergence of pancreatic cell lineages in the embryo through the lens of cellular plasticity, emphasizing how reciprocal interactions with the microenvironment influence lineage decisions. We also draw parallels between developmental and pathological processes, highlighting the dual role of plasticity: enabling lineage specification during development and supporting regenerative responses upon injury or in disease states. A deeper appreciation of the mechanisms underlying pancreatic cell identity and plasticity holds the potential to inform the development of new therapeutic modalities for pancreatic diseases, such as diabetes and pancreatic cancer.

Organ function depends on the precise spatial organization of cells across multiple scales, from individual units to cellular communities that form local niches and, ultimately, higher-order structures. Although cell identities are increasingly well defined, the spatial arrangement and interactions among diverse cell types remain poorly understood. Here, we combine single-cell and spatial transcriptomics to map pancreatic cell populations across space and time, from embryonic development to adult homeostasis in mice. Using these maps, we resolve spatial heterogeneity among pancreatic cell types and uncover epithelial-mesenchymal units as basic tissue niches, which we functionally characterize in both mouse and human models. We also demonstrate that the mesenchymal lineage diversifies into various specialized subtypes during development, but this complexity diminishes over time, ultimately converging into a few fibroblast subtypes in adulthood. Together, our findings reveal how different progenitor lineages codevelop and organize into structured communities that establish a functional pancreas, providing a framework to guide in vitro organogenesis and tissue engineering for pancreatic diseases.

The pancreas has vital endocrine and exocrine functions that can be affected by life-threatening diseases such as diabetes and pancreatic cancer. Although animal models are 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 essential for investigating pancreatic development and disease in the human context. However, the production of human stem cell-derived pancreatic organoids with a proper branched architecture and correct patterning of cell domains remains challenging. Here, we successfully developed a protocol that efficiently and reproducibly generated organoids from human induced pluripotent stem cells (hiPSCs) by optimizing organoid culture format and media. Our differentiation protocol promotes acinar cell differentiation and generates organoids with branches patterned into the 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 pancreatic development in a system that recapitulates the most important aspects of pancreatic tissue architecture. Lastly, we anticipate that this system will contribute to the replacement of animal models used to investigate diseases, such as pancreatic cancer.

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.

Cellular plasticity underpins heterogeneity in embryogenic progenitor cells and cancer cells. The transcription factor deltaNp63 ( ΔNp63 ) has been implicated in regulating cellular plasticity in several epithelial tissues. Despite a recently established role in steering plasticity of pancreatic cancer, ΔNp63 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 ΔNP63 in the embryonic pancreas. ΔNP63 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 ΔNP63 in knock-out mice leads to hypotrophic exocrine acini with reduced levels of differentiation markers. In summary, ΔNp63 confers heterogeneity within the MPP compartment, supporting exocrine cell development. These new insights in developmental plasticity have potential implications for pancreatic regeneration and cancer.

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.

In vitro models of the pancreas can aid in developing therapies for pancreatic diseases. Nonetheless, most pancreatic tissue engineering is limited to insulin‐secreting β‐cells or pancreatic adenocarcinoma models. Combining all essential tissue components, including exocrine, endocrine, and blood vasculature, is crucial to recapitulate native tissue organization. In this study, extrusion‐based 3D bioprinting to create pancreatic tissue constructs containing both endocrine and exocrine compartments is exploited. Mouse pluripotent stem cell‐derived pancreatic progenitors, pancreatic endothelial cells, and mesenchymal stem cells are bioprinted. During postprinting cultivation, the cells differentiated into exocrine and endocrine lineages, resulting in vascularized pancreatic tissue‐like constructs with multiple cell types. However, the bioprinted constructs contracted significantly postprinting, hindering control of cell positioning and shape preservation. Therefore, 2 strategies to reduce the contraction and deformation of the bioprinted constructs are developed. These bioprinting techniques and biomaterial combinations allow us to investigate the influence of construct design and cellular composition on pancreatic cell fate. The results reveal that increased construct stiffness and endothelial component presence significantly promoted endocrine while suppressing exocrine differentiation. Overall, a novel strategy for pancreatic tissue engineering that advances and holds promise for pancreas disease and development modeling, as well as pharmaceutical testing is demonstrated.