Psoriatic disease initially affects the skin and later extends to the joints. Here, we show a two-step process that orchestrates the spread of inflammation from the skin to the joints. Induction of psoriatic skin disease in photoconvertible mice, followed by sequencing and computational characterization of skin-derived cells in the joints, was used to identify a population of CD2+MHC-II+CCR2+ myeloid precursors that builds a skin-derived myeloid cell compartment in the joints. Single-cell cross-species reference mapping and mitochondrial variant tracing showed an orthologous human cell population. Interactome analysis of the joints showed that in a second step, resident regulatory CD200+ fibroblasts regulate the priming of CD2+MHC-II+CCR2+ myeloid precursors, which subsequently control IL-17 expression in T cells. Hence, the spread of inflammation requires a distinct migratory myeloid precursor population and a permissive local tissue environment, similar to tumor metastasis.

Vaccines have greatly improved public health since their proper establishment, but there are fundamental disconnects in the way that vaccines are developed, namely from cells and animals, then to humans, leading to their failure in final clinical trials. Fortunately, organoids, especially immune and tumor organoids, can replicate sophisticated insights regarding human tissue architecture and functionality in organ-like 3D structures, thereby emerging as a powerful tool to bridge the gap between animals and humans. In this review, we first highlight the mechanisms of vaccine-induced protection, then discuss the development of immune and tumor organoids as novel tools for vaccine testing, design, and reverse translation. We also indicate specific hurdles facing the application of organoids in vaccinology and offer forward-looking perspectives.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic inflammation and progressive damage to connective tissue. It is driven by dysregulated cellular homeostasis, often leading to autoimmune destruction and permanent disability in severe cases. Over the past decade, various drug delivery systems have been developed to enable targeted therapies for disease prevention, reduction, or suppression. As an emerging therapeutic platform, extracellular vesicles (EVs) offer several advantages over conventional drug delivery systems, including biocompatibility and low immunogenicity. Consequently, an increasing number of studies have explored EV-based delivery systems in the treatment of RA, leveraging their natural ability to evade phagocytosis, prolong in vivo half-life, and minimize the immunogenicity of therapeutic agents. In this review, we first provide an in-depth overview of the pathogenesis of RA and the current treatment landscape. We then discuss the classification and biological properties of EVs, their potential therapeutic mechanisms, and the latest advancements in EVs as drug delivery platforms for RA therapy. We emphasize the significance of EVs as carriers in RA treatment and their potential to revolutionize therapeutic strategies. Furthermore, we examine key technological innovations and the future trajectory of EV research, focusing on the challenges and opportunities in translating these platforms into clinical practice. Our discussion aims to offer a comprehensive understanding of the current state and future prospects of EV-based therapeutics in RA.

Psoriatic disease initially affects the skin, but later extends to the joints. Herein, we describe a two-step process that orchestrates spreading of inflammation from the skin to the joints. Induction of psoriatic skin disease in photoconvertible mice, followed by sequencing and computational characterization of skin-derived cells in the joints, identified a unique population of CD2+ MHC-II+ CCR2+ myeloid precursors that built up a skin-derived myeloid cell compartment in the joints. Single-cell cross-species reference mapping and mitochondrial variant tracing showed an orthologue human cell population. Interactome analyses in the joints showed that in a second step, resident regulatory CD200+ fibroblasts critically regulate the priming of the CD2+ MHC-II+ CCR2+ myeloid precursors, which subsequently regulate the IL-17 expression in T cells. Hence, spreading of inflammation requires a distinct migratory myeloid precursor population and a permissive local tissue environment, similar to tumour metastasis. ![Figure][1] ### Competing Interest Statement Peter Rhein is employed by Cytek Biosciences, the maker of the Amnis brand ImageStream, which was used in this study. Oliver Distler has/had consultancy relationship with and/or has received research funding from and/or has served as a speaker for the following companies in the area of rheumatology in the last three calendar years: 4P-Pharma, Abbvie, Acepodia, Aera, AnaMar, Anaveon, Argenx, AstraZeneca, Boehringer Ingelheim, BMS, Calluna, Cantargia AB, CSL Behring, EMD Serono, Galderma, Galapagos, Gossamer, Hemetron, Innovaderm, Janssen, Lilly, MSD Merck, Nkarta, Novartis, Oorja Bio, Orion, Pilan, Prometheus, Quell, Redxpharma, Scleroderma Research Foundation, Sumitomo, Topadur, UCB and Umlaut bio. Patent issued 'mir-29 for the treatment of systemic sclerosis' (US8247389, EP2331143). Co-founder of CITUS AG. The rest of the authors declare no competing interests. [1]: pending:yes

The placenta forms the maternal-fetal interface to protect the developing fetus from xenobiotics or pathogens. However, the understanding of complex placental features and responses to pathogens are hindered due to the lack of near-physiological models. Here, we present an engineered human placental organoid microphysiological system (MPS) incorporated with vascular endothelium, which allows to recapitulate early placental features in a vascular niche. The MPS comprises a customized insert-based organ chip and a rocker, enabling in situ differentiation and formation of placental organoids from human trophoblast stem cells under dynamic culture conditions. By incorporating vascular endothelium, trophoblast organoids (TOs) maintain improved cell viability, long-term trophoblast proliferation and differentiation. Moreover, trophoblast organoids cocultured with endothelium (EndTOs) show the activation of innate immune-related signaling pathways and high-level secretion of distinct immunomodulatory factors, including antiviral type I and III interferons and trophoblast-specific factors. We further demonstrate that EndTOs exhibit attenuated susceptibility to Zika virus (ZIKV) than single cultured TOs, indicating the crucial role of vascular niche in enhancing intrinsic antiviral defenses functions of trophoblasts. This bioinspired placental organoid MPS provides a useful platform for studying placental physiology and relevant diseases.

Fibroblasts are important regulators of inflammation, but whether fibroblasts change phenotype during resolution of inflammation is not clear. Here we use positron emission tomography to detect fibroblast activation protein (FAP) as a means to visualize fibroblast activation in vivo during inflammation in humans. While tracer accumulation is high in active arthritis, it decreases after tumor necrosis factor and interleukin-17A inhibition. Biopsy-based single-cell RNA-sequencing analyses in experimental arthritis show that FAP signal reduction reflects a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalization) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalization). Spatial transcriptomics of human joints indicates that pro-resolving niches of CD200+DKK3+ fibroblasts cluster with type 2 innate lymphoid cells, whereas MMP3+/IL6+ fibroblasts colocalize with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilized the type 2 innate lymphoid cell phenotype and induced resolution of arthritis via CD200-CD200R1 signaling. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

Organoids-on-chips is an emerging innovative integration of stem cell-derived organoids with advanced organ-on-chip technology, providing a novel platform for the in vitro construction of biomimetic micro-physiological systems. The synergistic merger transcends the limitations of traditional drug screening and safety assessment methodologies, such as 2D cell cultures and animal models. In this review, we examine the prevailing challenges and prerequisites of preclinical models utilized for drug screening and safety evaluations. We highlighted the salient features and merits of organoids-on-chip, elucidating their capability to authentically replicate human physiology, thereby addressing contemporary impediments. We comprehensively overviewed the recent endeavors where organoids-on-chips have been harnessed for drug screening and safety assessment and delved into potential opportunities and challenges for evolving sophisticated, near-physiological organoids-on-chips. Based on current achievements, we further discuss how to enhance the practicality of organoids-on-chips and accelerate the translation from preclinical to clinical stages in healthcare and industry by utilizing multidisciplinary convergent innovation.

Brain organoids hold great potential for modeling human brain development and pathogenesis. They recapitulate certain aspects of the transcriptional trajectory, cellular diversity, tissue architecture and functions of the developing brain. In this review, we explore the engineering strategies to control the molecular-, cellular- and tissue-level inputs to achieve high-fidelity brain organoids. We review the application of brain organoids in neural disorder modeling and emerging bioengineering methods to improve data collection and feature extraction at multiscale. The integration of multiscale engineering strategies and analytical methods has significant potential to advance insight into neurological disorders and accelerate drug development.

Understanding the impact of long-term opioid exposure on the embryonic brain is critical due to the surging number of pregnant mothers with opioid dependency. However, this has been limited by human brain inaccessibility and cross-species differences in animal models. Here, a human midbrain model is established that uses hiPSC-derived midbrain organoids to assess cell-type-specific responses to acute and chronic fentanyl treatment and fentanyl withdrawal. Single-cell mRNA sequencing of 25,510 cells from organoids in different treatment groups reveals that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters synaptic activity and neuron projection. In contrast, acute fentanyl treatment increases dopamine release but does not significantly alter gene expression related to cell lineage development. These results provide the first examination of the effects of opioid exposure on human midbrain development at the single-cell level.

Light sheet fluorescence microscopy (LSFM) is a widely used imaging technique for living and large cleared samples. However, high-performance LSFM systems are often prohibitively expensive and not easily scalable for high-throughput applications. Here, we introduce a cost-effective, scalable, and versatile high-resolution imaging framework, called projected Light Sheet Microscopy (pLSM), which repurposes readily available off-the-shelf consumer-grade components and an over-the-network control architecture to achieve high-resolution imaging of living and cleared samples. We extensively characterize the pLSM framework and showcase its capabilities through high-resolution, multi-color imaging and quantitative analysis of mouse and post-mortem human brain samples cleared using various techniques. Moreover, we show the applicability of pLSM for high-throughput molecular phenotyping of human induced pluripotent cells (iPSC)-derived brain and vessel organoids. Additionally, we utilized pLSM for comprehensive live imaging of bacterial pellicle biofilms at the air-liquid interface, uncovering their intricate layered architecture and diverse cellular dynamics across different depths. Overall, the pLSM framework has the potential to further democratize LSFM by making high-resolution light sheet microscopy more accessible and scalable.

Vascular malformation, a key clinical phenotype of Proteus syndrome, lacks effective models for pathophysiological study and drug development due to limited patient sample access. To bridge this gap, we built a human vascular organoid model replicating Proteus syndrome’s vasculature. Using CRISPR/Cas9 genome editing and gene overexpression, we created induced pluripotent stem cells (iPSCs) embodying the Proteus syndrome-specific AKT E17K point mutation for organoid generation. Our findings revealed that AKT overactivation in these organoids resulted in smaller sizes yet increased vascular connectivity, although with less stable connections. This could be due to the significant vasculogenesis induced by AKT overactivation. This phenomenon likely stems from boosted vasculogenesis triggered by AKT overactivation, leading to increased vascular sprouting. Additionally, a notable increase in dysfunctional PDGFRβ + mural cells, impaired in matrix secretion, was observed in these AKT-overactivated organoids. The application of AKT inhibitors (ARQ092, AZD5363, or GDC0068) reversed the vascular malformations; the inhibitors’ effectiveness was directly linked to reduced connectivity in the organoids. In summary, our study introduces an innovative in vitro model combining organoid technology and gene editing to explore vascular pathophysiology in Proteus syndrome. This model not only simulates Proteus syndrome vasculature but also holds potential for mimicking vasculatures of other genetically driven diseases. It represents an advance in drug development for rare diseases, historically plagued by slow progress.

Abstract Fibroblasts are key orchestrators of inflammation. Little is known whether these cells change phenotype during resolution of inflammation. We adopted a method to visualise fibroblast activation during inflammation in humans in vivo, which is based on a fibroblast activation protein (FAP) tracer detected by positron emission tomography (PET). While tracer accumulation was high in active arthritis, it decreased significantly after TNF- and IL-17A inhibition. Biopsy-based scRNA-seq analyses in experimental arthritis demonstrated that FAP signal reduction reflected a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalisation) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalisation). Spatial transcriptomics of human joints revealed that pro-resolving niches of CD200+DKK3+ fibroblasts clustered with innate lymphoid cells (ILC)2, whereas MMP3+/IL6+ fibroblasts were co-localised with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilised the ILC2 phenotype and induced resolution of arthritis via CD200/CD200R1 pathway. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

Effective delivery of the CRISPR-Cas9 components is crucial to realizing the therapeutic potential. Although many delivery approaches have been developed for this application, oral delivery has not been explored due to the degradative nature of the gastrointestinal tract. For this issue, we developed a series of novel phenylboronic acid (PBA)-functionalized chitosan-polyethylenimine (CS-PEI) polymers for oral CRISPR delivery. PBA functionalization equipped the polyplex with higher stability, smooth transport across the mucus, and efficient endosomal escape and cytosolic unpackaging in the cells. From a library of 12 PBA-functionalized CS-PEI polyplexes, we identified a formulation that showed the most effective penetration in the intestinal mucosa after oral gavage to mice. The optimized formulation performed feasible CRISPR-mediated downregulation of the target protein and reduction in the downstream cholesterol. As the first oral CRISPR carrier, this study suggests the potential of addressing the needs of both local and systemic editing in a patient-compliant manner.

ABSTRACT Joint fibroblasts play an important role in the transition from joint inflammation to irreversible joint damage. There is no established clinical method to measure fibroblast activation during inflammation and their phenotypic dynamics upon therapy to date. Here we show that upon treatment with IL-17A/TNF-blocking antibodies fibroblasts change their phenotype from a destructive IL-6 + /MMP3 + THY1 + to a CD200 + DKK3 + subtype, actively inducing resolution of inflammation. This phenotypic switch can be visualized due to so far unexplored different capacities of fibroblast subtypes with regard to receptor internalization of small molecular tracers with high affinity to FAP. Although FAP expression levels are comparable between fibroblast subtypes in the joint, FAP internalisation rate correlates with the destructive potential of fibroblasts and resolving fibroblasts have a lower FAP internalisation rate, providing a valuable imaging tool to visualize the transition from joint damage to resolution of inflammation.

Bioengineered strategies enable gut chips to faithfully replicate essential features of intestinal microsystems, encompassing geometric properties, peristalsis, intraluminal fluid flow, oxygen gradients, and the microbiome. This emerging technique serves as a powerful tool for nutrition studies by emulating the absorption and distribution processes in a manner highly relevant to human physiology. It offers unprecedented accessibility for investigating the mechanisms governing nutrition metabolism. While the application of gut-on-chip models in disease modeling and drug screening has been extensively explored, their potential in dietary nutrition research remains relatively unexplored. This comprehensive review provides an overview of the different approaches employed in constructing gut-on-chip platforms using diverse cell sources and niche mimics. Furthermore, it explores the applications and prospects of gut-on-chips in nutrition-related investigations, with a specific focus on carotenoid transport, absorption, and metabolism. Lastly, this review discusses the future development trajectory of this groundbreaking technology paradigm, highlighting its broad applicability in the field of food technology. By harnessing the capabilities of these state-of-the-art techniques within gut chip platforms, researchers can establish a robust scientific foundation for unraveling the intricate mechanisms that govern the behavior and functional properties of carotenoids.

The impact of long-term opioid exposure on the embryonic brain is crucial to healthcare due to the surging number of pregnant mothers with an opioid dependency. Current studies on the neuronal effects are limited due to human brain inaccessibility and cross-species differences among animal models. Here, we report a model to assess cell-type specific responses to acute and chronic fentanyl treatment, as well as fentanyl withdrawal, using human induced pluripotent stem cell (hiPSC)-derived midbrain organoids. Single cell mRNA sequencing (25,510 single cells in total) results suggest that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters the pathways associated with synaptic activities and neuron projection. Acute fentanyl treatment, however, increases dopamine release but does not induce significant changes in gene expressions of cell lineage development. To date, our study is the first unbiased examination of midbrain transcriptomics with synthetic opioid treatment at the single cell level.

DiGeorge Syndrome, or 22q11.2 deletion syndrome (22q11.2 DS), is a genetic disorder caused by microdeletions in chromosome 22, impairing the function of endothelial cells (EC) and/or mural cells and leading to deficits in blood vessel development such as abnormal aortic arch morphology, tortuous retinal vessels, and tetralogy of Fallot. The mechanism by which dysfunctional endothelial cells and pericytes contribute to the vasculopathy, however, remains unknown. In this study, we used human blood vessel organoids (VOs) generated from iPSC of 22q11.2 DS patients to model the vascular malformations and genetic dysfunctions. We combined high-resolution lightsheet imaging and single-cell transcriptome analysis to link the genetic profile and vascular phenotype at the single-cell level. We developed a comprehensive analytical methodology by integrating deep learning-mediated blood vessel segmentation, network graph construction, and tessellation analysis for automated morphology characterization. We report that 22q11.2DS VOs demonstrate a smaller size with increased angiogenesis/sprouting, suggesting a less stable vascular network. Overall, clinical presentations of smaller vascular diameter, less connected vasculature, and increased branch points were recapitulated in 22q11.2DS VOs. Single-cell transcriptome profiling showed heterogeneity in both 22q11.2DS and control VOs, but the former demonstrated alterations in endothelial characteristics that are organ-specific and suggest a perturbation in the vascular developmental process. Intercellular communication analysis indicated that the vascular dysfunctions in 22q11.2 deletion were due to a lower cell-cell contact and upregulated extracellular matrix organization involving collagen and fibronectin. Voronoi diagram-based tessellation analysis also indicated that the colocalization of endothelial tubes and mural cells was different between control and 22q11.2 VOs, indicating that alterations in EC and mural interactions might contribute to the deficits in vascular network formation. This study illustrates the utility of VO in revealing the pathogenesis of 22q11.2DS vasculopathy.

Immune-mediated inflammatory diseases (IMIDs), such as inflammatory bowel diseases and inflammatory arthritis (e.g., rheumatoid arthritis, psoriatic arthritis), are marked by increasing worldwide incidence rates. Apart from irreversible damage of the affected tissue, the systemic nature of these diseases heightens the incidence of cardiovascular insults and colitis-associated neoplasia. Only 40–60% of patients respond to currently used standard-of-care immunotherapies. In addition to this limited long-term effectiveness, all current therapies have to be given on a lifelong basis as they are unable to specifically reprogram the inflammatory process and thus achieve a true cure of the disease. On the other hand, the development of various OMICs technologies is considered as “the great hope” for improving the treatment of IMIDs. This review sheds light on the progressive development and the numerous approaches from basic science that gradually lead to the transfer from “bench to bedside” and the implementation into general patient care procedures.