Establishment and characterization of turtle liver organoids provides a potential model to decode their unique adaptations

Painted turtles are remarkable for their freeze tolerance and supercooling ability along with their associated resilience to hypoxia/anoxia and oxidative stress, rendering them an ideal biomedical model for hypoxia-induced injuries (including strokes), tissue cooling during surgeries, and organ cryopreservation. Yet, such research is hindered by their seasonal reproduction and slow maturation. Here we developed and characterized adult stem cell-derived turtle liver organoids (3D self-assembled in vitro structures) from painted, snapping, and spiny softshell turtles spanning ~175My of evolution, with a subset cryopreserved. This development is, to the best of our knowledge, a first for this vertebrate Order, and complements the only other non-avian reptile organoids from snake venom glands. Preliminary characterization, including morphological, transcriptomic, and proteomic analyses, revealed organoids enriched in cholangiocytes. Deriving organoids from distant turtles and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently lacking in herpetological research. Such platform could potentially support studies including genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, with implications for ecological, evolutionary, and biomedical research

Derivation and characterization of novel organoid lines for use in biomedical research

The advent of adult-stem cell derived organoids have unlocked the potential for a variety of new use cases to emerge in the biomedical and basic biology fields of research. The expansion of new and useful organoid models continues to emerge. Here we derived and completed in-depth characterization of multiple canine organoid lines in CHAPTER 2. CHAPTER 3 explores the ability to genetically modify these canine hepatic organoids by using CRISPR/Cas9 and an HDR template to create an exact mutation found in collies. CHAPTER 4 focuses on the expansion of use cases for the use of the canine colonoid model by assessing its ability to respond to inflammation and further suppression using a corticosteroid on the 2D Transwell system. Finally, CHAPTER 5, the first turtle-derived hepatic organoids were isolated, expanded, and further characterized using a variety of techniques and approaches. These various characterized organoid lines are preserved in a biobank and allow for future investigations in biomedical research for both human and veterinary investigators

Leveraging the predictive power of 3D organoids in dogs to develop new treatments for man and man’s best friend

Recent progress in adult stem cell technology, along with interdisciplinary collaboration in the field of One Health, has accelerated the development of 3D organoid cultures in non-model animals, such as dogs. These advancements have the potential to significantly impact disease modeling and drug development for many diseases shared between man and man’s best friend

Culture and characterization of canine and feline corneal epithelial organoids: A new tool for the study and treatment of corneal diseases

In this study, we isolated and cultured canine and feline 3D corneal organoids. Samples derived from corneal limbal epithelium from one canine and one feline patient were obtained by enucleation after euthanasia. Stem cell isolation and organoid culture were performed by culturing organoids in Matrigel. Organoids were subsequently embedded in paraffin for further characterization. The expression of key corneal epithelial and stromal cell markers in canine and feline organoids was evaluated at the mRNA level by RNA-ISH and at the protein level by immunofluorescence (IF) and immunohistochemistry (IHC), while histochemical analysis was performed on both tissues and organoids using periodic-acid Schiff (PAS), Sirius Red, Gomori's Trichrome, and Colloidal Iron stains. IF showed consistent expression of AQP1 within canine and feline organoids and tissues. P63 was present in canine tissues, canine organoids, and feline tissues, but not in feline organoids. Results from IHC staining further confirmed the primarily epithelial origin of the organoids. Canine and feline 3D corneal organoids can successfully be cultured and maintained and express epithelial and stem cell progenitor markers typical of the cornea. This novel in vitro model can be used in veterinary ophthalmology disease modeling, corneal drug testing, and regenerative medicine.This article is published as Bedos, Leila, Hannah Wickham, Vojtech Gabriel, Christopher Zdyrski, Rachel A. Allbaugh, Dipak Kumar Sahoo, Lionel Sebbag, Jonathan P. Mochel, and Karin Allenspach. "Culture and characterization of canine and feline corneal epithelial organoids: A new tool for the study and treatment of corneal diseases." Frontiers in Veterinary Science 9 (2022): 1050467. DOI: 10.3389/fvets.2022.1050467. Copyright 2022 Bedos, Wickham, Gabriel, Zdyrski, Allbaugh, Sahoo, Sebbag, Mochel and Allenspach. Attribution 4.0 International (CC BY 4.0). Posted with permission

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability. Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells. The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol describes the tasks directly preceding an experiment, including in vitro validation of experimental results. Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.This article is published as Gabriel, Vojtech, Christopher Zdyrski, Dipak K. Sahoo, Kimberly Dao, Agnes Bourgois-Mochel, Todd Atherly, Marilyn N. Martinez et al. "Canine Intestinal Organoids in a Dual-Chamber Permeable Support System." Journal of Visualized Experiments: Jove 181 (2022). DOI: 10.3791/63612. Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted

Canine Intestinal Organoids as a Novel In Vitro Model of Intestinal Drug Permeability: A Proof-of-Concept Study

A key component of efforts to identify the biological and drug-specific aspects that contribute to therapeutic failure or unexpected exposure-associated toxicity is the study of drug-intestinal barrier interactions. While methods supporting such assessments are widely described for human therapeutics, there is relatively little information available for similar evaluation in support of veterinary pharmaceuticals. There is, therefore, a critical need to develop novel approaches for evaluating drug-gut interactions in veterinary medicine. Three-dimensional (3D) organoids can address these difficulties in a reasonably affordable system that circumvents the need for more invasive in vivo assays in live animals. However, a first step in the development of such systems is understanding organoid interactions in a 2D monolayer. Given the importance of orally administered medications for meeting the therapeutic need of companion animals, we demonstrate growth conditions under which canine colonoid-derived intestinal epithelial cells survive, mature, and differentiate into confluent cell systems with high monolayer integrity. We further examine the applicability of this canine colonoid-derived 2D model to assess the permeability of three structurally diverse, passively absorbed β-blockers (e.g., propranolol, metoprolol, and atenolol). Both absorptive and secretive apparent permeability (Papp) of these drugs at two different pH conditions were evaluated in canine colonoid-derived monolayers and were compared with that of Caco-2 cells. This proof-of-concept study provides promising preliminary results with regard to the utility of canine-derived organoid monolayers for species-specific assessments of therapeutic drug passive permeability.This is a pre-print of the article Sahoo, Dipak Kumar, Marilyn Martinez, Kimberly Dao, Vojtech Gabriel, Christopher Zdyrski, Albert E. Jergens, Todd Atherly et al. "Canine Intestinal Organoids as a Novel In Vitro Model of Intestinal Drug Permeability: A Proof-of-Concept Study." (2023): 2023040101. DOI: 10.20944/preprints202304.0101.v1. Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted

Changes of Enterocyte Morphology and Enterocyte: Goblet Cell Ratios in Dogs with Protein-Losing and Non-Protein-Losing Chronic Enteropathies

This study aimed to assess the morphometry of enterocytes as well as the goblet cell-to-enterocyte ratio in different intestinal segments of dogs with chronic enteropathies (CE). Histopathological intestinal samples from 97 dogs were included in the study (19 healthy juveniles, 21 healthy adults, 24 dogs with protein-losing enteropathy (PLE), and 33 CE dogs without PLE). Healthy adult small intestinal enterocytes showed progressively reduced epithelial cell height in the aboral direction, while juvenile dogs showed progressively increased epithelial cell height in the aboral direction. CE dogs had increased epithelial cell height in the duodenum, while PLE dogs had decreased epithelial cell heights compared to healthy adult dogs. Both the CE and PLE dogs showed decreased enterocyte width in the duodenal segment, and the ileal and colonic enterocytes of CE dogs were narrower than those of healthy adult dogs. CE dogs had a lower goblet cell-to-enterocyte ratio in the colon segment compared to healthy dogs. This study provides valuable morphometric information on enterocytes during canine chronic enteropathies, highlighting significant morphological enterocyte alterations, particularly in the small intestine, as well as a reduced goblet cell-to-enterocyte ratio in the colon of CE cases compared to healthy adult dogs.This article is published as Díaz-Regañón D, Gabriel V, Livania V, Liu D, Ahmed BH, Lincoln A, Wickham H, Ralston A, Merodio MM, Sahoo DK, et al. Changes of Enterocyte Morphology and Enterocyte: Goblet Cell Ratios in Dogs with Protein-Losing and Non-Protein-Losing Chronic Enteropathies. Veterinary Sciences. 2023; 10(7):417. https://doi.org/10.3390/vetsci10070417. Posted with permission.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

Canine Intestinal Organoids as a Novel In Vitro Model of Intestinal Drug Permeability: A Proof-of-Concept Study

A key component of efforts to identify the biological and drug-specific aspects contributing to therapeutic failure or unexpected exposure-associated toxicity is the study of drug–intestinal barrier interactions. While methods supporting such assessments are widely described for human therapeutics, relatively little information is available for similar evaluations in support of veterinary pharmaceuticals. There is, therefore, a critical need to develop novel approaches for evaluating drug–gut interactions in veterinary medicine. Three-dimensional (3D) organoids can address these difficulties in a reasonably affordable system that circumvents the need for more invasive in vivo assays in live animals. However, a first step in developing such systems is understanding organoid interactions in a 2D monolayer. Given the importance of orally administered medications for meeting the therapeutic need of companion animals, we demonstrate growth conditions under which canine-colonoid-derived intestinal epithelial cells survive, mature, and differentiate into confluent cell systems with high monolayer integrity. We further examine the applicability of this canine-colonoid-derived 2D model to assess the permeability of three structurally diverse, passively absorbed β-blockers (e.g., propranolol, metoprolol, and atenolol). Both the absorptive and secretive apparent permeability (Papp) of these drugs at two different pH conditions were evaluated in canine-colonoid-derived monolayers and compared with that of Caco-2 cells. This proof-of-concept study provides promising preliminary results with regard to the utility of canine-derived organoid monolayers for species-specific assessments of therapeutic drug passive permeability

Characterization of the First Turtle Organoids: A Model for Investigating Unique Adaptations with Biomedical Potential

Painted turtles are remarkable for their well-developed freeze tolerance and associated resilience to hypoxia/anoxia, oxidative stress, and ability to supercool. They are, therefore, an ideal model for biomedical research on hypoxia-induced injuries (including strokes), tissue cooling during extensive surgeries, and organ cryopreservation. Yet, the seasonal reproduction and slow maturation of turtles hinder basic and applied biomedical research. To overcome these limitations, we developed the first adult stem cell-derived turtle hepatic organoids, which provide 3D self-assembled structures that mimic their original tissue and allow for in vitro testing and experimentation without constantly harvesting donor tissue and screening offspring. Our pioneering work with turtles represents the first for this vertebrate Order and complements the only other organoid lines from non-avian reptiles, derived from snake venom glands. Here we report the isolation and characterization of hepatic organoids derived from painted, snapping, and spiny softshell turtles spanning ∼175 million years of evolution, with a subset being preserved in a biobank. Morphological and transcriptomics revealed organoid cells resembling cholangiocytes, which was then compared to the tissue of origin. Deriving turtle organoids from multiple species and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently missing in most herpetological research. When combined with genetic editing, this platform will further support studies of genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, among others. We discuss the unique abilities of turtles, including their overwintering potential, which has implications for ecological, evolutionary, and biomedical research.This is a pre-print of the article Zdyrski, Christopher, Vojtech Gabriel, Thea B. Gessler, Abigail Ralston, Itzel Sifuentes-Romero, Debosmita Kundu, Sydney Honold et al. "Characterization of the First Turtle Organoids: A Model for Investigating Unique Adaptations with Biomedical Potential." bioRxiv (2023): 2023-02. DOI: 10.1101/2023.02.20.527070. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Copyright 2023. The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Posted with permission

Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organs epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine