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Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

Here, we describe the generation of floating cultures of apical-out intestinal organoids from hydrogel-embedded intestinal organoid cultures. ...

University of Veterinary Medicine

Recent technical advances in the stem cell field have enabled the in vitro generation of complex structures resembling whole organs termed organoids. Most of these approaches employ culture systems that allow stem cell-derived or tissue progenitor cells to self-organize into three-dimensional (3D)-structures. Since organoids can be grown from different species (human, mouse, cat, dog), organs (intestine, kidney, brain, liver), and from patient-derived induced pluripotent stem cells, they create significant prospects for modelling development and diseases, for toxicology and drug discovery studies, and in the field of regenerative medicine. Here, we report on intestinal stem cells, organoid culture, organoid disease modeling, transplantation, specifically covering the current and future uses of this exciting new insight model to the field of veterinary medicine.

Intestinal Organoids-Current and Future Applications.

Scientific information on spontaneous type I diabetes mellitus (DM) and treatment modalities in guinea pigs is scarce. As most diabetic guinea pigs are overweight and respond to dietary changes, a disorder resembling type II-DM in humans seems to be most prevalent in this species. In the present report, a nine-month-old female intact guinea pig (GP1) was presented because of a cataract and polyphagia. The physical examinations in GP1 and its littermate, GP2, were unremarkable. Laboratory tests revealed hyperglycemia, hyperlipidemia, elevated fructosamine concentrations, and glucosuria in GP1 and GP2. Not responding to dietary changes, an insulin-dependent diabetes mellitus was suspected in both animals. Treatment with 0.5 IU of glargine insulin (Lantus) per guinea pig subcutaneously (s.c.) once daily was initiated in both animals. Monitoring included repeated clinical evaluations and the measurement of plasma glucose and fructosamine concentrations. Capillary glucose concentration was measured using a glucometer, and glucosuria was monitored by dipstick. Blood glucose concentrations decreased quickly in both GPs, and glucosuria resolved. Including several dose adjustments, DM remained controlled for over 1.5 years. Bilateral cataracts and lens-induced uveitis in GP1 were medically managed with only slight progression. This is the first report of guinea pigs with insulin-dependent diabetes mellitus that were successfully treated with long-acting basal insulin glargine.

Successful Insulin Glargine Treatment in Two Pet Guinea Pigs with Suspected Type 1 Diabetes Mellitus.

In contrast to humans and dogs, the skin microbiota of wolves is yet to be described. Here, we investigated the skin microbiota of dogs and wolves kept in outdoor packs at the Wolf Science Center (WSC) via 16S rRNA gene amplicon sequencing. Skin swab samples were also collected from human care takers and their pet dogs. When comparing the three canine groups, representing different degrees of human contact to the care takers and each other, the pet dogs showed the highest level of diversity. Additionally, while human skin was dominated by a few abundant phylotypes, the skin microbiota of the care takers who had particularly close contact with the WSC animals was more similar to the microbiota of dogs and wolves compared to the humans who had less contact with these animals. Our results suggest that domestication may have an impact on the diversity of the skin microbiota, and that the canine skin microbiota can be shared with humans, depending on the level of interaction.

Wolves, dogs and humans in regular contact can mutually impact each other's skin microbiota.

To compare the effects of open-tube blood sampling with previously investigated blood sampling methods via evacuated tube on thromboelastography variables for blood samples from dogs.

Comparison of the effects of open-tube and evacuated tube-assisted sampling methods on thromboelastography variables for blood samples from healthy dogs.

The role of the gut microbiome in developing Inflammatory Bowel Disease (IBD) in humans and dogs has received attention in recent years. Evidence suggests that IBD is associated with alterations in gut microbial composition, but further research is needed in veterinary medicine. The impact of IBD treatment on the gut microbiome needs to be better understood, especially in a breed-specific form of IBD in Yorkshire Terriers known as Yorkshire Terrier Enteropathy (YTE). This study aimed to investigate the difference in gut microbiome composition between YTE dogs during disease and remission and healthy Yorkshire Terriers. Our results showed a significant increase in specific taxa such as Clostridium sensu stricto 1, Escherichia-Shigella, and Streptococcus, and a decrease in Bacteroides, Prevotella, Alloprevotella, and Phascolarctobacterium in YTE dogs compared to healthy controls. No significant difference was found between the microbiome of dogs in remission and those with active disease, suggesting that the gut microbiome is affected beyond clinical recovery.

Gut microbiome signatures of Yorkshire Terrier enteropathy during disease and remission.

Glycemic variability (GV) refers to swings in blood glucose levels and is an emerging measure of glycemic control in clinical practice. It is associated with micro- and macrovascular complications and poor clinical outcomes in diabetic humans. Although an integral part of patient assessment in human patients, it is to a large extent neglected in insulin-treated diabetic dogs. This prospective pilot study was performed to describe canine within-day GV in non-diabetic dogs with the aim to provide a basis for the interpretation of daily glucose profiles, and to promote GV as an accessible tool for future studies in veterinary medicine. Interstitial glucose concentrations of ten non-diabetic, non-obese beagles were continuously measured over a 48-h period using a flash glucose monitoring system. GV was assessed using the common indices MAGE (mean amplitude of glycemic excursion), GVP (Glycemic variability percentage) and CV (coefficient of variation). A total of 2260 sensor measurements were obtained, ranging from 3.7 mmol/L (67 mg/dL) to 8.5 mmol/L (153 mg/dL). Glucose profiles suggested a meal-dependent circadian rhythmicity with small but significant surges during the feeding periods. No differences in GV indices were observed between day and night periods (p > 0.05). The MAGE (mmol/L), GVP (%) and CV (%) were 0.86 (± 0.19), 7.37 (± 1.65), 6.72 (± 0.89) on day one, and 0.83 (± 0.18), 6.95 (± 1.52), 6.72 (± 1.53) on day two, respectively. The results of this study suggest that GV is low in non-diabetic dogs and that glucose concentrations are kept within narrow ranges.

Short-term glycemic variability in non-diabetic, non-obese dogs assessed by common glycemic variability indices.

Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.

Iwan Anton Burgener

Small Animal Internal Medicine,
Department for Small Animals and Horses,
Small Animal Internal Medicine, Department for Small Animals and Horses

University of Veterinary Medicine

Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

Iwan Anton Burgener

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Small Animal Internal Medicine,Department for Small Animals and Horses,Small Animal Internal Medicine, Department for Small Animals and Horses

University of Veterinary Medicine

Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

Small Animal Internal Medicine,
Department for Small Animals and Horses,
Small Animal Internal Medicine, Department for Small Animals and Horses