Pathophysiological Research Research Articles

Emerging Trends in Pathophysiology: Insights from the 9th International Congress of the International Society for Pathophysiology (ISP 2023).

This article provides a summary of the 9th International Congress of the International Society for Pathophysiology (ISP 2023) which took place in Belgrade, Serbia, from 4 to 6 July 2023. It describes the main trends and the most promising areas of research in current pathophysiology, including investigations of new pathogenic pathways, and the identification of cellular and molecular mechanisms, target molecules, genetic mechanisms, and new therapeutic strategies. The present article also highlights the research conducted by leading scientific teams and national pathophysiological societies from various countries that adds to our understanding of the pathogenesis of diseases and pathological processes.

Therapeutics in cystic fibrosis: Clinical revolution and new challenges

Over time, cystic fibrosis has become a model of synergy between research in pathophysiology and cell biology, and clinical advances. Therapies targeting the CFTR protein, in particular CFTR modulators, have transformed the prognosis of patients, bringing the hope of a normal life with the possibility of starting a family and growing old, challenging established statistics. However, patients are not yet cured, and side effects remain insufficiently documented. Epidemiological changes create new challenges for the management of cystic fibrosis. Approximately 10 % of patients still lack a therapeutic option. The community of researchers, pharmaceutical industries, patient associations, and health authorities remains committed to monitor the long-term effects of these still poorly characterised treatments, and to explore new pharmacological approaches, such as gene therapies.

Ultrasound diagnostics in solving situational problems in the discipline of clinical pathophysiology

Aim. Тo train future specialists to effectively apply the methods of pathophysiological research (ultrasound diagnostics) in solving situational problems within the course of clinical pathophysiology.Materials and methods. The 2nd and 3rd year students were trained to use stationary and portable ultrasound machines Phillips CX50 and Chison Sonotouch 80. The students were trained to master ultrasound B and M modes, Doppler ultrasonography, as well as elastography of the most common diseases of the endocrine, digestive, and urinary systems. At the first stage, students worked in tandem with the lecture, observing the process of sensor manipulation. At the second stage, each student conducted a study on his or her fellow student.Results. When conducting ultrasound scanning of the hepatoduodenal zone and kidneys, 65% and 55% of students, respectively, failed to obtain an optimal image and to measure anatomical structures during the assigned time interval. Echocardioscopy was established to be the most difficult method to master when solving situational problems within the topic of “Impaired Systemic Circulation”. This task was successfully performed only by 20% of the students. The optimal student-lecturer ratio was established to be 6:1.Conclusion. The use of new ultrasound technologies in the training process is important for forming students’ professional competencies and increasing their motivation.

Bibliometric analysis of myelin imaging studies of patients with multiple sclerosis (2000-2022).

Multiple sclerosis (MS) is a condition that can impact the central nervous system (CNS) and cause damage to the myelin, which is responsible for facilitating the normal transmission of electrical impulses along the nerves. We performed a bibliometric analysis of the scientific publications on myelin imaging in MS to reveal the development trends in this field and to evaluate research trends in myelin imaging in MS. The Web of Science Core Collection was searched for articles related to myelin imaging in MS published between January 2000 and December 2022. CiteSpace, VOSviewer, and R language were used to evaluate and visualize contributions by and co-occurrence relationships among countries and institutions, authors, journals, citations, keywords, and so on. A total of 1,639 articles addressed the topic of myelin imaging in MS. The United States had the largest number of annual publications. The University of London was the institution with the highest number of publications (n=118) and citations (n=9,885). The top 3 productive authors were all from the University of British Columbia in Canada. An article published by Mackay et al. in 1994 had the most citations (n=272). Neuroimage [impact factor (IF) =7.40, Journal Citation Reports quartile 1 (Q1)] was the most productive journal in terms of the number of articles relating to myelin imaging in MS (n=149). In recent years, myelin water imaging, synthetic magnetic resonance imaging (SyMRI), inhomogeneous magnetization, positron emission tomography (PET) imaging, and aquaporin-4 (AQP4) have been researched hotspots of myelin imaging in MS. With advancements in the pathophysiological research on myelin changes in MS, myelin imaging is playing an important role in the diagnosis and treatment of MS. In addition, the use of new sequences of myelin imaging to distinguish MS from other inflammatory demyelinating diseases is a future development trend in this field.

In Vitro Culture of White Adipose Tissue.

White adipose tissue (WAT) plays a crucial endocrine organ that regulates blood glucose and lipid levels, satiety, and inflammation. Before the described technique, primary white adipocytes could not be stably cultured in vitro. The lack of a reliable primary culture model impeded research in WAT metabolism and drug development. We have developed a novel technique for WAT primary culture called "sandwiched white adipose tissue" (SWAT). SWAT overcomes the natural buoyancy of adipocytes by sandwiching minced WAT between sheets of adipose-derived stromal cells. The resulting constructs are viable for at least 8weeks in culture. SWAT maintains the intact extracellular matrix, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

Progress in pathophysiology research and update on diagnosis and treatment strategies for fecal incontinence

The traditional barrier theory believes that fecal incontinence is related to an imbalance of the recto-anal barrier and the characteristics of stool. However, in clinical practice this theory proves unable to explain all types of fecal incontinence. In recent years, research on the mechanisms related to fecal incontinence has shifted to a new integrative concept with the rectum and anus as functional units, and the central-peripheral nervous system and internal and external anal sphincters as a control loop. The diagnosis and treatment strategy of fecal incontinence, which is replaced by sacral neuromodulation, is undergoing a quiet change. With the progressively aging population in China, the need to improve the diagnosis and treatment of fecal incontinence has become increasingly urgent. This article explores the trends in diagnosis and treatment and mechanism research from the perspective of recent advances in pathophysiological research and updated diagnosis and treatment methods for fecal incontinence.

Extremes of baseline cognitive function determine the severity and outcomes of delirium: findings from a prospective longitudinal cohort study

AbstractBackgroundEpidemiological studies have shown the bidirectional relationship between dementia and delirium to be complex and non‐linear. This has implications for mechanistic studies investigating the extent to which any pathophysiology might be specific to delirium, or shared with dementia. While delirium is associated with long‐term dementia risk and cognitive decline, this association may be independent of classical Alzheimer’s pathology in oldest‐old patients.MethodThe Delirium and Population Health informatics (DELPHIC) study prospectively followed a population‐representative sample of 1510 individuals aged ≥70 years living in London, UK. We performed cognitive assessments at baseline, two years, and ascertained delirium symptoms during incident hospitalisation in 209 participants across 371 episodes.ResultBetter baseline cognitive function was generally associated with a lower likelihood of delirium. However, during delirium episodes, there was a U‐shaped relationship between baseline cognition and delirium symptom severity; participants with the lowest and highest baseline cognition experienced the most severe delirium and arousal deficits (baseline z‐score ‐2: mean MDAS 15; baseline z‐score +2: mean MDAS 11). At follow‐up, we observed the largest cognitive decline in participants with better baseline cognition experiencing delirium (2.2SD decline vs 0.8SD without delirium).ConclusionThese findings imply that delirium pathophysiology research should take into account differences in baseline cognition, for example by defining pre‐existing subclinical dementia pathology. We suggest the need for prospective, serial pathophysiological studies to consider the temporal sequence of delirium‐related neural damage. In this way, we could differentiate which mechanisms are unique to delirium, and which might interact with pre‐existing dementia pathophysiology to result in long‐term cognitive deficits.

Association between inflammatory bowel disease and subsequent depression or anxiety disorders - A retrospective cohort study of 31,728 outpatients

BackgroundCrohn's disease (CD) and ulcerative colitis (UC) are characterized by chronic intestinal and systemic inflammation. The extraintestinal sequelae of inflammatory bowel disease (IBD) are major contributors to disease morbidity and significantly affect patients' quality of life. Here, we evaluated the association between IBD and subsequent depression or anxiety disorder in a large outpatient collective from Germany. Methods15,864 individual IBD patients (CD: n = 6,791, UC: n = 9073) and 15,864 nearest neighbor propensity score matched patients without IBD were included from the Disease Analyzer database (IQVIA). Diagnoses of depression and anxiety disorders were compared between IBD and non-IBD patients during a five-year follow-up period using Kaplan-Meier estimators and Cox-regression models. ResultsAfter 5 years of follow-up, depression was diagnosed in 14.4% of CD patients versus 10.2% of matched pairs (p < 0.001) and in 13.1% of UC patients versus 10.1% of matched pairs (p < 0.001). In line, the incidence of anxiety order was significantly higher among CD (4.7% vs. 4.4%, p = 0.009) and UC patients (4.3% vs. 3.5%, p = 0.005). Regression analysis confirmed a significant association between IBD and both mental conditions (Hazard Ratio (HR)CD/depression: 1.40, HRUC/depression: 1.32, HRCD/anxiety disorder: 1.21, HRUC/anxiety disorder: 1.28). Subgroup analyses revealed a stronger association for CD and depression (HR: 1.51) and UC and depression (HR:1.49) among male patients as well as UC and anxiety disorders (HR: 1.51) among female patients. ConclusionOur data argue for a significant association between IBD and mental diseases including depression and anxiety disorders. Although further pathophysiological research is warranted, we hypothesize that specific psychological screening measures in IBD patients could improve quality of life and outcome.

Non-Arteritic Anterior Ischemic Optic Neuropathy (NA-AION): A Comprehensive Overview.

Non-arteritic anterior ischemic optic neuropathy (NA-AION) represents one of the most important causes of blindness or severely impaired vision in middle-aged and elderly people. Unilateral optic disc edema and abrupt, painless vision loss are its defining features. It is commonly assumed that NA-AION is caused by an ischemic infarction of the optic nerve head, and, although the exact pathogenesis is still unknown, several risk factors and comorbidities associated with its development have been found. NA-AION occurs generally in patients older than 50 years who have small optic discs and vasculopathy risk factors. Even though numerous treatment options have been proposed, no available effective medical or surgical therapy or prophylactic measure for NA-AION currently exists. The purpose of present-day therapeutic strategies is therefore to identify and possibly control any underlying modifiable risk factors, aiming to prevent the development of new NA-AION episodes in the affected and fellow eye. A thorough assessment of NAION, including its history, epidemiology, etiology, pathophysiology, risk factors, associated comorbidities, clinical findings, diagnostic tests, treatment choices, prognosis, and future research, is the goal of this work.

Chitosan stabilized copper iodide nanoparticles enabled nano-bio-engineered platform for efficient electrochemical biosensing of dopamine

Neurodegenerative disorders are one of the significant challenges to the aging society, as per the United Nations, where 1 in 6 people globally over 65 years of age are expected to suffer by 2050. The exact pathophysiological root of these disorders is although not known adequately, but reduced dopamine (most significant neurotransmitters) levels have been reported in people affected by Parkinson's disease. Sensitive detection and effective monitoring of dopamine can help to diagnose these neurodegenerative disorders at a very early stage, which will help to properly treat these disorders and slow down their progression. Therefore, it is crucial to detect physiological and clinically acceptable amounts of dopamine with high sensitivity and selectivity in basic pathophysiology research, medication, and illness diagnosis. Here in this present investigation, nano-bio-engineered stable chitosan stabilized copper iodide nanoparticles (CS@CuI NPs) were synthesized to engineer the active biosensing platform for developing dopamine biosensors. Initially, the as-synthesized nano-bio-engineered CS@CuI NPs were subjected to its drop-casting onto an Indium tin oxide (ITO) conducting glass substrate. This substrate platform was then utilized to immobilize tyrosinase (Tyr) enzyme by drop-casting to fabricate Tyr/CS@CuI NPs/ITO bioelectrode for the ultrasensitive determination of dopamine. Several techniques were used to characterize the structural, optical, and morphological properties of the synthesized CS@CuI NPs and Tyr/CS@CuI NPs/ITO bioelectrode. Further, the as-prepared bioelectrode was evaluated for its suitability and electrocatalytic behaviour towards dopamine by cyclic voltammetry. A perusal of the electroanalytic results of the fabricated biosensor revealed that under the optimized experimental conditions, Tyr/CS@CuI NPs/ITO bioelectrode exhibits a very high electrochemical sensitivity of 11.64 μA μM−1 cm−2 towards dopamine with the low limit of detection and quantification of 0.02 and 0.386 μM, respectively. In addition, the fabricated bioelectrode was stable up to 46 days with only 4.82 % current loss, reusable till 20 scans, and it also performed effectively while real sample analysis. Therefore, the nano-bio-engineered biosensor platform being reported can determine deficient dopamine levels in a very selective and sensitive manner, which can help adequately manage neurodegenerative disorders, further slowing down the disease progression.

Compartmentalized primary cultures of dorsal root ganglion neurons to model peripheral pathophysiological conditions.

Neurosensory disorders such as pain and pruritus remain a major health problem greatly impacting the quality of life, and often increasing the risk of mortality. Current pre-clinical models to investigate dysfunction of sensory neurons have shown a limited clinical translation, in part, by failing to mimic the compartmentalized nociceptor anatomy that exhibit a central compartment containing the soma and a peripheral one harboring the axon endings with distinct molecular and cellular environmental composition. Thus, there is a need to validate compartmentalized preclinical neurosensory models for investigating the pathophysiology of peripheral sensory disorders and to test drug candidates. Here, we have addressed this issue and developed a microfluidic-based preclinical nociceptor model and validated it for investigating inflammatory and neuropathic peripheral disorders. We show that this model reproduces the peripheral sensitization and resolution produced by an inflammatory soup and by the chemotherapeutic drug paclitaxel. Furthermore, compartmentalized nociceptor primary cultures were amenable to co-culture with keratinocytes in the axonal compartment. Interaction of axonal endings with keratinocytes modulated neuronal responses, consistent with a crosstalk between both cell types. These findings pave the way towards translational pre-clinical sensory models for skin pathophysiological research and drug development.

Self-reported continuing professional development needs of medical laboratory professionals in Ghana

BackgroundBecause of the essential nature of the work of medical laboratory professionals, continuing development in knowledge and skills is indispensable. The study aimed at identifying and prioritizing the development and training needs of medical laboratory professionals in Ghana. This is expected to help in developing focused continuing professional development (CPD) that meets the needs of practitioners as well as the changing medical trends.MethodsAn online cross-sectional survey in February 2022 using a structured questionnaire was conducted. Respondents were asked questions that collected demographic and work-related data about them, their participation, preference, and challenges in being part of CPDs. Finally, a list of topics based on (i) quality management systems, (ii) technical competence, (iii) laboratory management, leadership, and coaching, (iv) pathophysiology, and (iv) data interpretation and research were asked with the option to rate them on a 3-point scale (most, moderate, and least) in order of importance.ResultsA total of 316 medical laboratory professionals participated in the study. Overall, the most frequently selected topics for training based on domains for CPD training and ranking as most important were (i) quality management systems, (mean = 80.59 ± 9.024; 95% CI = 73.04–88.13); (ii) pathophysiology, data interpretation, and research (mean = 78.0 ± 6.973; 95% CI = 73.97–82.03); (iii) technical competence (mean = 73.97 ± 10.65; 95% CI = 66.35–81.59); and (iv) laboratory management, leadership, and coaching (mean = 72.82 ± 9.719; 95% CI = 67.44–78.2). The factors affecting the choice of training needs included the medical laboratory professionals’ current place of work, years in service, the reason for attending CPD activities, the period for attending the last CPD, being in a supervisory role, and the number of staff being supervised. Face-to-face presentations, training workshops, and hands-on workshops were the most preferred modes of CPD delivery with financial implications and workload/time constraints being the main challenges impeding CPD participation.ConclusionThe identified needs will help in developing CPD programs that address what medical laboratory professionals prioritize as training needs. Stakeholders should incorporate these training needs into future programs and address the challenges highlighted in this study to have more relevant training for medical laboratory professionals.

Echocardiographic evaluation of cardiac reserve to detect subtle cardiac dysfunction in mice

AimsCardiac reserve is a sensitive tool for early detection of cardiac dysfunction. However, cardiac reserve assessment by catecholamine stress echocardiography in mice varied in the doses of β-adrenergic agonists and the time point for measurements, which may lead to inaccurate readouts. This study aims to establish a standardized protocol for assessing cardiac reserve in mice. Main methodsC57BL/6J mice under isoflurane anesthesia were intraperitoneally injected with varying doses of isoproterenol (Iso), and subjected to echocardiographic measurements. Key findingsHeart rate (HR), ejection fraction (EF), fractional shortening (FS), global longitudinal strain (GLS) and strain rate all reached peak values within 1–3 min after Iso injection at doses higher than 0.2 mg/kg. Compared with 0.1 mg/kg Iso, 0.2 mg/kg Iso resulted in higher HR, EF, FS and GLS, whereas doses higher than 0.2 mg/kg did not yield further increase. Cardiac response of female mice recapitulated main characteristics of those of male mice except that female mice displayed higher maximum HR and were more sensitive to higher doses of Iso. Furthermore, the advantages of present stress protocol over conventional baseline echocardiographic measurements were verified in comparisons of exercised vs. sedentary and aged vs. young mice for cardiac function evaluation. SignificanceWe developed a reproducible and sensitive approach to evaluate cardiac reserve by continuously monitoring cardiac function every minute for 3 min after 0.2 mg/kg Iso injection. This approach will enable detection of subtle cardiac dysfunction and accelerate innovative research in cardiac pathophysiology.

Should Ovarian Tissue Cryopreservation in Pediatric Patients with Turner Syndrome Be Limited to the Research Setting?

Now that ovarian tissue cryopreservation (OTC) has become standard of care for patients receiving gonadotoxic therapies, discussion has turned towards offering OTC to pediatric patients with Turner syndrome outside of research. While patients with Turner syndrome have unmet fertility needs and the authors support efforts for fertility preservation in these individuals, safety and efficacy data about OTC in this population are limited. Building on longstanding debates around offering experimental therapies as research or outside of research (as "innovative therapy"), we considered the suitability of offering OTC for Turner syndrome as innovative therapy. Based on pathophysiology and preliminary research data, we argue that there is significant uncertainty about whether the risk-benefit profile of OTC for Turner syndrome patients is favorable. This reduces the weight of arguments in favor of offering it as innovative therapy. Furthermore, as Turner syndrome is rare, widespread availability of OTC could make it difficult to develop generalizable knowledge. The benefits of innovative therapy for acquiring experience from use in humans and avoiding research-related procedures are of limited importance too, as OTC is already an established procedure and current studies involve limited procedures that restrict access. OTC should therefore only be offered to Turner syndrome patients in research settings, until additional data suggest the risk-benefit profile is likely favorable.

Diabetes as a consequence of acute pancreatitis.

Diabetes is a highly prevalent disease that was initially simplified into three major types: Type 1, type 2 and gestational diabetes. With the global rise in incidence of acute pancreatitis (AP), a lesser-known type of diabetes referred to as diabetes of the exocrine pancreas (DEP) is becoming more recognized. However, there is a poor understanding of the inherent relationship between diabetes and AP. There is established data about certain diseases affecting the exocrine function of the pancreas which can lead to diabetes. More specifically, there are well established guidelines for diagnosis and management of DEP caused be chronic pancreatitis. Conversely, the sequelae of AP leading to diabetes has limited recognition and data. The purpose of this review is to provide a comprehensive summary of the prevalence, epidemiology, pathophysiology and future research aims of AP-related diabetes. In addition, we propose a screening and diagnostic algorithm to aid clinicians in providing better care for their patients.

Staying Up to Date With Evolving Postpartum Depression Pathophysiology and Treatment Research.

Perinatal depression (PND) is one of the most common medical conditions associated with pregnancy, with 1 in 7 women impacted by PND symptoms and 1 in 13 meeting criteria for major depressive disorder. Unfortunately, half of postpartum depression (PPD) cases begin during pregnancy but are not diagnosed until postpartum. Delayed diagnosis and treatment of PND lead to poor outcomes for both mother and child. The American College of Obstetricians and Gynecologists recently updated its recommendation that screening for perinatal depression and anxiety occur at the initial prenatal visit, later in pregnancy, and at postpartum. Several hypotheses have been developed to explain the pathophysiology of PND including endocrine, epigenetic, synaptic transmission, neural network, neurosteroid, stress, and inflammatory mechanisms. Researchers believe that the answer lies in a synthesized mechanism of all of these models. Novel and emerging therapeutics are focusing on the neurosteroid mechanism within the integrated hypothesis. Neuroactive steroids are changing the understanding of the pathophysiology of depression and PPD, and novel and emerging therapeutics with new mechanisms of action based on these findings are impacting the treatment paradigm for this widespread and burdensome disorder.

A turn-on fluorescence strategy for hypochlorous acid detection based on DNAzyme-assisted cyclic signal amplification

Hypochlorous acid (HClO) is a crucial active oxygen component and one of the innate immunity's barrier substances in the body. Abnormal fluctuations in HClO concentration can lead to increased oxidative stress, cellular dysfunction, and the onset of various diseases. Thus, developing convenient, affordable, efficient, and sensitive methods to monitor HClO concentration in healthcare and pathophysiology research is highly significant. In this study, we developed a novel fluorescence strategy for HClO detection based on nucleic acid oxidative cleavage and Pb2+-dependent DNAzyme. By introducing a phosphorothioate site in the hairpin-structured nucleic acid sequence, the nucleic acid probe specifically recognized HClO and underwent oxidative cleavage. Upon cleavage, the enzyme strand is liberated, forming DNAzyme. This DNAzyme then cleaves the substrate strand, liberating the initially quenched fluorescent dyes and generating a turn-on fluorescent signal. The enzyme strand produced by the oxidative cleavage of HClO can be repeatedly utilized, thus realizing the cyclic signal amplification to reduce background noise. We verified the detection mechanism of this strategy through stepwise fluorescence spectroscopy analysis and electrophoresis. Under optimal experimental conditions, the method achieved a detection limit of 5.38 nM and a linear range of 1 nM-800 nM. This method demonstrated exceptional performance in actual biological sample testing and presented an exciting opportunity for expanded utilization in clinical diagnosis and medical research.

The Association Between Taxane Use and Lacrimal Disorders

Purpose The current study seeks to investigate the association between lacrimal disorders and the use of docetaxel and paclitaxel. Methods A disproportionality analysis was conducted using the United States FDA Adverse Event Reporting System (FAERS). All adverse event reports containing the term docetaxel or paclitaxel were selected. Lacrimal adverse events were identified using the lacrimal disorders Standardized MedDRA Query (SMQ), which includes disorders that affect lacrimal gland and drainage system including blockage of nasolacrimal duct, occlusion/stenosis of punctum, lacrimal gland neoplasms, and inflammations and infections. Results The proportionate reporting ratio (PRR) comparing lacrimal events among docetaxel to paclitaxel users was 2.47 (95% CI, 2.03–3.02). With respect to specific lacrimal events, dacryostenosis (PRR 19.54 [95% CI, 7.19–53.13]), increased lacrimation (PRR 3.2 [95% CI, 2.42–4.23]), lacrimation disorder (p = 0.02), and xeropthalmia reports (p > 0.001) were significantly more common. Conclusions The growing body of epidemiologic, clinical, and pathophysiologic research supports the case that docetaxel leads to adverse lacrimal events in certain patients and should be taken into consideration when oncologists consider docetaxel vs. paclitaxel.

Multiplex immunofluorescence staining of coverslip-mounted paraffin-embedded tissue sections.

Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin-embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re-staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal-like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin-embedded tissue was used for multiplex IF staining with increased Z-resolution. In summary, this method offers time-saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.

A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease.

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/β-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.