Organoids, combined with genetic editing strategies, have the potential to offer rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing ...efficiency of organoids with the use of non-viral electroporation methods has only been up to 30%, with implications for the subsequent need for selection, including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method for intestinal organoid editing using a ribonucleoprotein-based CRISPR approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples with no observed off-target editing. The method allowed us to study the effect of loss of the tumour suppressor gene PTEN in normal human intestinal cells. Analysis of PTEN-deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss, including mTORC1 activation.
Infectious diseases are increasingly recognized as a major threat worldwide due to the rise of antimicrobial resistance and the emergence of novel pathogens. In vitro models that can adequately mimic ...in vivo gastrointestinal physiology are in high demand to elucidate mechanisms behind pathogen infectivity, and to aid the design of effective preventive and therapeutic interventions. There exists a trade‐off between simple and high throughput models and those that are more complex and physiologically relevant. The complexity of the model used shall be guided by the biological question to be addressed. This review provides an overview of the structure and function of the intestine and the models that are developed to emulate this. Conventional models are discussed in addition to emerging models which employ engineering principles to equip them with necessary advanced monitoring capabilities for intestinal host‐pathogen interrogation. Limitations of current models and future perspectives on the field are presented.
Infectious diseases are increasingly recognized as a major threat worldwide due to the rise of antimicrobial resistance and the emergence of novel pathogens. In vitro models that can adequately mimic in vivo gastrointestinal physiology are thus in high demand. This review provides an overview of such models and their applicability for interrogating host‐pathogen interactions.
Summary
Purpose: Data on epileptiform electroencephalography (EEG) discharges in healthy children are limited, with published studies dating back more than 20 years. Moreover, analyses have been ...performed exclusively using paper‐recorded EEG, and reported prevalences differ significantly. With recent reports using these data as reference suggesting an increased prevalence of epileptiform EEG discharges in children with behavioral disturbances, acquisition of exact prevalence data has become even more critical. The aim of our study was to analyze the frequency of epileptiform EEG discharges in healthy children using digitally recorded EEG (DEEG) and to compare these data to those of previously published studies.
Methods: Prospective analysis of DEEG was performed in 382 healthy children (226 male, 156 female) ages 6–13 years admitted to our hospital for minor head trauma. Recording was carried out for a minimum of 20 min including hyperventilation and photic stimulation. Analysis was carried out by two board‐certified clinical neurophysiologists.
Results: Epileptiform EEG discharges were detected in 25 of 382 children (11 of 226 male, 14 of 156 female) corresponding to an overall prevalence of 6.5%. Of these 25 children, 4 had either generalized or bifrontal spikes, 12 showed constant localized focal discharges, and 9 showed multifocal discharges. Compared to previous studies using non‐DEEG recording, the prevalence of epileptiform EEG discharges in our population was significantly higher. No significant difference was found when comparing our data to prevalences recently reported in children with behavioral disturbances using DEEG.
Conclusions: Our study further highlights the urgent need to reevaluate the prevalence of epileptiform EEG discharges in healthy children using DEEG recordings in a large cohort.
Inflammatory bowel diseases IBD such as Crohn's disease CD and ulcerative colitis UC are complex conditions presenting with a wide range of phenotypes. Given major variation in disease severity and ...outcomes as well as response to existing therapies, a personalised treatment approach stands the chance of improving the overall disease outcome as well as minimising potentially harmful side effects. However, disease activity or distribution at the point of diagnosis are poor predictors of future disease outcome. Hence, the urgent need to develop biomarkers that could either predict the overall disease course i.e., disease prognostic biomarkers or the response to individual therapies i.e., disease predictive biomarkers. Despite the widely accepted need for such biomarkers to improve the management of IBD patients, their development has proven to be challenging for a number of reasons. Based on our own experience in this field, we perform a reality check on existing evidence, discuss main challenges, and outline future perspectives.
Here, we describe protocols for the preparation and dissociation of human fetal and pediatric intestinal tissue to a high-viability epithelial single-cell suspension. This epithelium-enriched ...single-cell suspension can then be used to generate single-cell RNA sequencing data as well as to create human intestinal organoids from both the fetal and pediatric intestine. Finally, this protocol details the dissociation of the intestinal organoids for use in single-cell analysis or passaging of organoids.
For complete details on the use and execution of this protocol, please refer to Elmentaite et al. (2020).
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•Dissection of the human embryonic/fetal intestine•Dissociation and enrichment of human intestinal epithelia from fetal/mucosal biopsies•Establishment and maintenance of intestinal organoids from single-cell suspension•Dissociation of organoids to single-cell solution
Here, we describe protocols for the preparation and dissociation of human fetal and pediatric intestinal tissue to a high-viability epithelial single-cell suspension. This epithelium-enriched single-cell suspension can then be used to generate single-cell RNA sequencing data as well as to create human intestinal organoids from both the fetal and pediatric intestine. Finally, this protocol details the dissociation of the intestinal organoids for use in single-cell analysis or passaging of organoids.
Epigenetic signatures are highly cell type specific. Separation of distinct cell populations is therefore desirable for all epigenetic studies. However, to date little information is available on ...whether separation protocols might influence epigenetic and/or gene expression signatures and hence might be less beneficial. We investigated the influence of two frequently used protocols to isolate intestinal epithelium cells (IECs) from 6 healthy individuals.
Epithelial cells were isolated from small bowel (i.e. terminal ileum) biopsies using EDTA/DTT and enzymatic release followed by magnetic bead sorting via EPCAM labeled microbeads. Effects on gene/mRNA expression were analyzed using a real time PCR based expression array. DNA methylation was assessed by pyrosequencing of bisulfite converted DNA and methylated DNA immunoprecipitation (MeDIP).
While cell purity was >95% using both cell separation approaches, gene expression analysis revealed significantly higher mRNA levels of several inflammatory genes in EDTA/DTT when compared to enzymatically released cells. In contrast, DNA methylation of selected genes was less variable and only revealed subtle differences. Comparison of DNA methylation of the epithelial cell marker EPCAM in unseparated whole biopsy samples with separated epithelium (i.e. EPCAM positive and negative fraction) demonstrated significant differences in DNA methylation between all three tissue fractions indicating cell type specific methylation patterns can be masked in unseparated tissue samples.
Taken together, our data highlight the importance of considering the potential effect of cell separation on gene expression as well as DNA methylation signatures. The decision to separate tissue samples will therefore depend on study design and specific separation protocols.
Abstract Background Currently there is much controversy whether to treat idiopathic facial palsy with corticosteroids with sparse data on the natural course of the disease in children. Methods We ...performed a prospective study on all children <15 years of age who were admitted to our unit for facial palsy between 1st July 1998 to 30th June 2008. All patients received a standardized work-up and follow-up. Therapy consisted of symptomatic treatment either with (in case of neuroborreliosis) or without a 14 day course of intravenous antibiotics. Findings 106 patients were included in our study. The calculated incidence for facial palsy was 21.1/100000/year for children <15 years. The incidence for neuroborreliosis (NB) in this age group was calculated to be 4.9/100000/year. The overall rate of complete recovery was 97.6% with significantly faster recovery in younger children and in patients with NB as compared to idiopathic facial palsy. Both patients with incomplete recovery were at least 14 years old and presented late in the course of the disease. Conclusion Based on the rate of 97.6% spontaneous complete recovery we believe that the routine use of corticosteroids in children with facial palsy is not justified, unless there is new data from controlled trials in children.
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