Organoids as regenerative medicine for inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic disorder with an increasing global prevalence. Managing disease activity relies on various pharmacological options. However, the effectiveness of current therapeutics is limited and not universally applicable to all patients and circumstances. Consequently, developing new management strategies is necessary. Recent advances in endoscopically obtained intestinal biopsy specimens have highlighted the potential of intestinal epithelial organoid transplantation as a novel therapeutic approach. Experimental studies using murine and human organoid transplantations have shown promising outcomes, including tissue regeneration and functional recovery. Human trials with organoid therapy have commenced; thus, this article provides readers with insights into the necessity and potential of intestinal organoid transplantation as a new regenerative therapeutic option in clinical settings and explores its associated challenges.

TGF-ß modulates cell fate in human ES cell-derived foregut endoderm by inhibiting Wnt and BMP signaling.

Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenous factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that transforming growth factor ß1 (TGF-ß1) activates a putative human OTX2/LHX1 gene regulatory network which promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, TGF-ß1 effects cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors render TGF-ß1-treated cells refractory to Wnt signaling. Subsequently, TGF-ß1-mediated inhibition of BMP and Wnt signaling suppresses liver fate and promotes pancreas fate. Furthermore, combined TGF-ß1 treatment and Wnt inhibition during pancreatic specification reproducibly and robustly enhance INSULIN+ cell yield across hESC lines. This modification of widely used differentiation protocols will enhance pancreatic ß cell yield for cell-based therapeutic applications.

Influence of vitamin D receptor signaling and vitamin D on colonic epithelial cell fate decisions in ulcerative colitis.

BACKGROUND AND AIMS: Epidemiological studies have shown that subnormal levels of vitamin D (25(OH)D) are associated with a more aggravated clinical course of ulcerative colitis (UC). Despite an increased focus on the therapeutic importance of vitamin D and vitamin D receptor (VDR) signaling, the mechanisms underlying the effects of the vitamin D-VDR axis on UC remain elusive. Therefore, we aimed to investigate whether exposure to active vitamin D (1,25(OH)2D3)/VDR signaling in human organoids could influence the maintenance of the colonic epithelium. METHODS: Intestinal VDR expression was studied by immunohistochemistry, RNA expression arrays, and single-cell RNA sequencing of colonic biopsy specimens obtained from patients with UC and healthy individuals. To characterize the functional and transcriptional effects of 1,25(OH)2D3, we used patient-derived colonic organoids. The dependency of VDR was assessed by knocking out the receptor with CRISPR/Cas9. RESULTS: Our results suggest that 1,25(OH)2D3/VDR stimulation supports differentiation of the colonic epithelium and that impaired 1,25(OH)2D3/VDR signaling thereby may compromise the structure of the intestinal epithelial barrier, leading to flares of UC. Furthermore, a transcriptional response to VDR activity was observed primarily in fully differentiated cells at the top of the colonic crypt, and this response was reduced during flares of UC. CONCLUSIONS: We identified an important role of vitamin D signaling in supporting differentiated cell states in the human colonic epithelium, and thereby maintenance of the intestinal barrier integrity. This makes the vitamin D-VDR signaling axis an interesting target for therapeutic efforts to achieve and maintain remission in patients with UC.

Patients' experience of a novel interdisciplinary nurse-led self-management intervention (INSELMA)-a qualitative evaluation.

BACKGROUND: Despite continuous improvements in anti-rheumatic pharmacological treatment, people with chronic inflammatory arthritis still report substantial disease impact. Based on the framework for complex interventions, we thus developed INSELMA, a novel nurse-coordinated multidisciplinary self-management intervention for patients with rheumatoid arthritis, psoriatic arthritis or axial spondyloarthritis. Based on individual biopsychosocial assessments, a rheumatology nurse facilitated goal setting and coordinated interdisciplinary support. The aim of this study was to explore the patients' experience of participating in the six-months INSELMA intervention. METHODS: Individual semi-structured interviews were conducted with 15 of the participants after their final follow-up. Thematic analysis was applied. RESULTS: The analysis derived four overall themes. (1) A new opportunity at the right time. The participants' disease impacted all areas of daily life. Participation in INSELMA was experienced as an opportunity to improve symptoms and together reduce long-held challenges they had fought alone, until now. (2) The importance of person-centred goals. The participants found it meaningful to work with their individual goals, which encompassed physical, psychological, and social factors. Having time between consultations to work with goals at home was important. (3) Empathy, partnership and a little nudging from health professionals are essential. The empathic nurses' continuous support and coaching helped participants become aware of their own resources. The participants highlighted having access to support from a physiotherapist and occupational therapist with rheumatology experience as important. (4) I got more than I could have hoped for. Most of the participants experienced decreased symptom load and improvement in physical strength, mobility, sleep, and mood as well as increased energy, knowledge, and self-management ability. The participants expressed new hope for the future with an improved ability to manage their symptoms and work towards new goals. CONCLUSION: The participants found the INSELMA intervention meaningful and feasible. They experienced decreased disease impact and increased activity levels, facilitated by empathy and self-management support from health professionals.

Immune cell-derived signals governing epithelial phenotypes in homeostasis and inflammation.

The intestinal epithelium fulfills important physiological functions and forms a physical barrier to the intestinal lumen. Barrier function is regulated by several pathways, and its impairment contributes to the pathogenesis of inflammatory bowel disease (IBD), a chronic inflammatory condition affecting more than seven million people worldwide. Current treatment options specifically target inflammatory mediators and have led to improvement of clinical outcomes; however, a significant proportion of patients experience treatment failure. Pro-repair effects of inflammatory mediators on the epithelium are emerging. In this review we summarize current knowledge on involved epithelial pathways, identify open questions, and put recent findings into clinical perspective, and pro-repair effects. A detailed understanding of epithelial pathways integrating mucosal stimuli in homeostasis and inflammation is crucial for the development of novel, more targeted therapies.

Development of a complex Interdisciplinary Nurse-coordinated SELf-MAnagement (INSELMA) intervention for patients with inflammatory arthritis.

BACKGROUND: Apart from a consistent focus on treating inflammation, patients with inflammatory arthritis (IA) report a range of unmet needs. Many experience not only residual symptoms but also various other physical, psychological, and social effects. Therefore, this study aimed to develop a complex Interdisciplinary Nurse-coordinated self-management (INSELMA) intervention for patients with IA, as an add-on treatment to usual outpatient care for those with substantial disease impact. METHODS: This study followed the British Medical Research Council's updated framework for developing complex interventions. The process encompassed the following steps: (1) The evidence base was identified; (2) workshops were held, involving 38 relevant stakeholders (managers, physicians, nurses, physiotherapists, occupational therapists, social workers, psychologists from hospitals and municipalities, and two patient research partners), to discuss and further develop the preliminary ideas; (3) relevant theories were identified (i.e., self-efficacy, acceptance and commitment therapy, and health literacy); (4) the intervention was modeled and remodeled and (5) the results, describing the final INSELMA intervention and outcomes. RESULTS: The INSELMA intervention encompasses an initial biopsychosocial assessment, which is performed by a rheumatology nurse. Then, activities that the participant wishes to improve are identified and goals are set. The nurse refers the participant to a multidisciplinary team and coordinates their support and relevant services in the participant's municipality. In addition, the health professionals have the opportunity to hold two interdisciplinary conferences during the intervention period. The participant and the health professionals work to achieve the set goals during a 6-month period, which ends with a status assessment and a discussion of further needs. The INSELMA intervention aims to increase self-management, reduce the impact of IA (e.g., pain, fatigue, sleep problems, and absenteeism), and increase self-efficacy, quality of life, mental well-being, work ability, and physical activity. CONCLUSIONS: The development of the INSELMA intervention involved stakeholders from two Danish rheumatology outpatient clinics, patient research partners and municipalities. We believe that we have identified important mechanisms to increase the self-management and quality of life of people with IA and to decrease the disease impact in those who are substantially affected. The health professionals involved have developed competences in delivering the intervention and it is ready to be tested in a feasibility study.

Detecting host responses to microbial stimulation using primary epithelial organoids.

The intestinal epithelium is constantly exposed to microbes residing in the lumen. Traditionally, the response to microbial interactions has been studied in cell lines derived from cancerous tissues, e.g. Caco-2. It is, however, unclear how the responses in these cancer cell lines reflect the responses of a normal epithelium and whether there might be microbial strain-specific effects. To address these questions, we derived organoids from the small intestine from a cohort of healthy individuals. Culturing intestinal epithelium on a flat laminin matrix induced their differentiation, facilitating analysis of microbial responses via the apical membrane normally exposed to the luminal content. Here, it was evident that the healthy epithelium across multiple individuals (n = 9) demonstrates robust acute both common and strain-specific responses to a range of probiotic bacterial strains (BB-12Ⓡ, LGGⓇ, DSM33361, and Bif195). Importantly, parallel experiments using the Caco-2 cell line provide no acute response. Collectively, we demonstrate that primary epithelial cells maintained as organoids represent a valuable resource for assessing interactions between the epithelium and luminal microbes across individuals, and that these models are likely to contribute to a better understanding of host microbe interactions.

Rapid Evolutionary Adaptation to Diet Composition in the Black Soldier Fly (Hermetia illucens).

Genetic adaptation of Hermetia illucens (BSF) to suboptimal single sourced waste streams can open new perspectives for insect production. Here, four BSF lines were maintained on a single sourced, low-quality wheat bran diet (WB) or on a high-quality chicken feed diet (CF) for 13 generations. We continuously evaluated presumed evolutionary responses in several performance traits to rearing on the two diets. Subsequently, we tested responses to interchanged diets, i.e., of larvae that had been reared on low-quality feed and tested on high-quality feed and vice versa to evaluate costs associated with adaptation to different diets. BSF were found to experience rapid adaptation to the diet composition. While performances on the WB diet were always inferior to the CF diet, the adaptive responses were stronger to the former diet. This stronger response was likely due to stronger selection pressure experienced by BSF fed on the low-quality single sourced diet. The interchanged diet experiment found no costs associated with diet adaptation, but revealed cross generational gain associated with the parental CF diet treatment. Our results revealed that BSF can rapidly respond adaptively to diet, although the mechanisms are yet to be determined. This has potential to be utilized in commercial insect breeding to produce lines tailored to specific diets.

ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

An organoid-based CRISPR-Cas9 screen for regulators of intestinal epithelial maturation and cell fate.

Generation of functionally mature organs requires exquisite control of transcriptional programs governing cell state transitions during development. Despite advances in understanding the behavior of adult intestinal stem cells and their progeny, the transcriptional regulators that control the emergence of the mature intestinal phenotype remain largely unknown. Using mouse fetal and adult small intestinal organoids, we uncover transcriptional differences between the fetal and adult state and identify rare adult-like cells present in fetal organoids. This suggests that fetal organoids have an inherent potential to mature, which is locked by a regulatory program. By implementing a CRISPR-Cas9 screen targeting transcriptional regulators expressed in fetal organoids, we establish Smarca4 and Smarcc1 as important factors safeguarding the immature progenitor state. Our approach demonstrates the utility of organoid models in the identification of factors regulating cell fate and state transitions during tissue maturation and reveals that SMARCA4 and SMARCC1 prevent precocious differentiation during intestinal development.

Transcriptional and epigenomic profiling identifies YAP signaling as a key regulator of intestinal epithelium maturation.

During intestinal organogenesis, equipotent epithelial progenitors mature into phenotypically distinct stem cells that are responsible for lifelong maintenance of the tissue. While the morphological changes associated with the transition are well characterized, the molecular mechanisms underpinning the maturation process are not fully understood. Here, we leverage intestinal organoid cultures to profile transcriptional, chromatin accessibility, DNA methylation, and three-dimensional (3D) chromatin conformation landscapes in fetal and adult epithelial cells. We observed prominent differences in gene expression and enhancer activity, which are accompanied by local changes in 3D organization, DNA accessibility, and methylation between the two cellular states. Using integrative analyses, we identified sustained Yes-Associated Protein (YAP) transcriptional activity as a major gatekeeper of the immature fetal state. We found the YAP-associated transcriptional network to be regulated at various levels of chromatin organization and likely to be coordinated by changes in extracellular matrix composition. Together, our work highlights the value of unbiased profiling of regulatory landscapes for the identification of key mechanisms underlying tissue maturation.

Organoids are not organs: Sources of variation and misinformation in organoid biology.

In the past decade, the term organoid has moved from obscurity to common use to describe a 3D in vitro cellular model of a tissue that recapitulates structural and functional elements of the in vivo organ it models. The term organoid is now applied to structures formed as a result of two distinct processes: the capacity for adult epithelial stem cells to re-create a tissue niche in vitro and the ability to direct the differentiation of pluripotent stem cells to a 3D self-organizing multicellular model of organogenesis. While these two organoid fields rely upon different stem cell types and recapitulate different processes, both share common challenges around robustness, accuracy, and reproducibility. Critically, organoids are not organs. This commentary serves to discuss these challenges, how they impact genuine utility, and shine a light on the need to improve the standards applied to all organoid approaches.

Maintenance of high-turnover tissues during and beyond homeostasis.

Tissues with a high turnover rate produce millions of cells daily and have abundant regenerative capacity. At the core of their maintenance are populations of stem cells that balance self-renewal and differentiation to produce the adequate numbers of specialized cells required for carrying out essential tissue functions. Here, we compare and contrast the intricate mechanisms and elements of homeostasis and injury-driven regeneration in the epidermis, hematopoietic system, and intestinal epithelium-the fastest renewing tissues in mammals. We highlight the functional relevance of the main mechanisms and identify open questions in the field of tissue maintenance.

Surfactant-Free Colloidal Syntheses of Gold-Based Nanomaterials in Alkaline Water and Mono-alcohol Mixtures.

Gold nanoparticles (Au NPs) and gold-based nanomaterials combine unique properties relevant for medicine, imaging, optics, sensing, catalysis, and energy conversion. While the Turkevich-Frens and Brust-Schiffrin methods remain the state-of-the-art colloidal syntheses of Au NPs, there is a need for more sustainable and tractable synthetic strategies leading to new model systems. In particular, stabilizers are almost systematically used in colloidal syntheses, but they can be detrimental for fundamental and applied studies. Here, a surfactant-free synthesis of size-controlled colloidal Au NPs stable for months is achieved by the simple reduction of HAuCl4 at room temperature in alkaline solutions of low-viscosity mono-alcohols such as ethanol or methanol and water, without the need for any other additives. Palladium (Pd) and bimetallic Au x Pd y NPs, nanocomposites and multimetallic samples, are also obtained and are readily active (electro)catalysts. The multiple benefits over the state-of-the-art syntheses that this simple synthesis bears for fundamental and applied research are highlighted.

Correlations between experiments and simulations for formic acid oxidation.

Electrocatalytic conversion of formic acid oxidation to CO2 and the related CO2 reduction to formic acid represent a potential closed carbon-loop based on renewable energy. However, formic acid fuel cells are inhibited by the formation of site-blocking species during the formic acid oxidation reaction. Recent studies have elucidated how the binding of carbon and hydrogen on catalyst surfaces promote CO2 reduction towards CO and formic acid. This has also given fundamental insights into the reverse reaction, i.e. the oxidation of formic acid. In this work, simulations on multiple materials have been combined with formic acid oxidation experiments on electrocatalysts to shed light on the reaction and the accompanying catalytic limitations. We correlate data on different catalysts to show that (i) formate, which is the proposed formic acid oxidation intermediate, has similar binding energetics on Pt, Pd and Ag, while Ag does not work as a catalyst, and (ii) *H adsorbed on the surface results in *CO formation and poisoning through a chemical disproportionation step. Using these results, the fundamental limitations can be revealed and progress our understanding of the mechanism of the formic acid oxidation reaction.

Ovarian apoptosis is regulated by carbohydrate intake but not by protein intake in speckled cockroaches.

When the likelihood of reproducing successfully is low, any prior investment in developing oocytes may be wasted. One means of recouping this investment is oosorption - where ova are absorbed and resources salvaged so they can be re-allocated to other traits. Food-limited female speckled cockroaches (Nauphoeta cinerea) appear to use this strategy. However, it is unclear if total food intake or the availability of specific nutrients induces this process. Here, we used the geometric framework of nutrition to determine how protein, carbohydrate and energy intake affect levels of ovarian apoptosis and necrosis (controlled versus uncontrolled cell death) in the terminal oocytes of female N. cinerea. We then compare the effects of nutrient intake on apoptosis (a key step towards oosorption) and offspring production to better understand the relationship between diet, apoptosis and female fitness. We found that even when food was abundant, females experienced high levels of apoptosis if their diet lacked carbohydrate. Necrosis was reduced when energy intake was high, but largely irrespective of nutrient ratio. Offspring production peaked on a low protein, high carbohydrate nutrient ratio (1P:7.96C), similar to that which minimized apoptosis (1P:7.34C) but not in the region of nutrient space that minimized necrosis. Thus, females consuming an ideal nutrient blend for reproduction can invest heavily in their current brood without needing to salvage nutrients from developing ova. However, offspring production was more dependent on carbohydrate consumption than apoptosis was, suggesting that the importance of carbohydrate in reproduction goes beyond regulating oosorption. This reliance on carbohydrate for female reproduction may reflect the unusual reproductive and nutritional physiology of speckled cockroaches; attributes that make this species an exciting model for understanding how diet regulates reproduction.

Robust Dual Optical Sensor for pH and Dissolved Oxygen.

As part of moving our optical pH and dissolved oxygen (DO) optical chemosensors toward industrial applications, we decided to explore a many-sensors-in-one principle. It was tested if physical segregation of the optical sensor components in a single sensor polymer could remove cross-talk and quenching. It was found that a design concept with an oxygen-responsive dye in polymer nanoparticles and a pH-responsive dye in an organically modified siloxane polymer resulted in a robust pH/O2 dual optical sensor. Individually, the O2-sensitive nanoparticles, a known component for optical DO sensing, and the pH sensor are operational. Thus, it was decided to test if nanoparticles enclosed within the pH-sensitive responsive sol-gel (i) could work together if segregated and (ii) could operate with a single intensity signal that is without a reference signal; developments within industrial optical sensor technology indicate that this should be feasible. The prototype optode produced in this work was shown to have a negligible drift over 60 h, bulk diffusion-limited DO response, and independent response to pH and O2. On the individual optode, pH calibration was found to show the expected sigmoidal shape and pKa, while the complexity of the calibration function for the DO signal was significant. While the engineering of the sensor device, optics, and hardware are not robust enough to attempt generic sensor calibration, it was decided to demonstrate the design concept in simple fermentation experiments. We conclude that the dual sensor design with the physical segregation of components is viable.

Molecular Manipulations and Intestinal Stem Cell-Derived Organoids in Inflammatory Bowel Disease.

The pathogenesis of inflammatory bowel diseases (IBD) involves genetic predisposition, environmental factors, and a broadly dysregulated intestinal immune response to the commensal intestinal microflora. The interface between genetic predisposition and environmental factors is reflected in the epigenetic regulation at the transcriptional level. Treatment targets now involve mucosal and histological healing, but the future might additionally include normalization of intestinal cellular functions also at the molecular level, for example comprising complete restoration of phenotypic, genotypic, and epigenetic states. Recent developments in patient-derived epithelial intestinal stem cell (ISC) organoid technologies have opened exciting new therapeutic opportunities to potentially attain molecular healing by combining stem cell therapy with molecular manipulations using (epi)drugs and/or CRISPR/Cas9 genome editing. Here, we are the first to discuss the possibility for phenotypic, genotypic, and epigenetic restoration via molecular manipulations and stem cell therapy in IBD from a clinical perspective.