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 organ's 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.

Impact of Environmental Risk Factors on Mitochondrial Dysfunction, Neuroinflammation, Protein Misfolding, and Oxidative Stress in the Etiopathogenesis of Parkinson's Disease.

As a prevalent progressive neurodegenerative disorder, Parkinson's disease (PD) is characterized by the neuropathological hallmark of the loss of nigrostriatal dopaminergic (DAergic) innervation and the appearance of Lewy bodies with aggregated α-synuclein. Although several familial forms of PD have been reported to be associated with several gene variants, most cases in nature are sporadic, triggered by a complex interplay of genetic and environmental risk factors. Numerous epidemiological studies during the past two decades have shown positive associations between PD and several environmental factors, including exposure to neurotoxic pesticides/herbicides and heavy metals as well as traumatic brain injury. Other environmental factors that have been implicated as potential risk factors for PD include industrial chemicals, wood pulp mills, farming, well-water consumption, and rural residence. In this review, we summarize the environmental toxicology of PD with the focus on the elaboration of chemical toxicity and the underlying pathogenic mechanisms associated with exposure to several neurotoxic chemicals, specifically 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat (PQ), dichloro-diphenyl-trichloroethane (DDT), dieldrin, manganese (Mn), and vanadium (V). Our overview of the current findings from cellular, animal, and human studies of PD provides information for possible intervention strategies aimed at halting the initiation and exacerbation of environmentally linked PD.

Cisplatin-Induced Metabolic Responses Measured with Raman Spectroscopy in Cancer Cells, Spheroids, and Canine-Derived Organoids.

Ex vivo assessment of drug response with conventional cell viability assays remains the standard practice for guiding initial therapeutic choices. However, such ensemble approaches fail to capture heterogeneities in treatment response and cannot identify early markers of response. Here, we leverage Raman spectroscopy (RS) as an accurate, low-cost, extraction-free, and label-free approach to track metabolic changes in cancer cells, spheroids, and organoids in response to cisplatin treatment. We identified 12 statistically significant metabolites in cells and 19 metabolites in spheroids and organoids as a function of depth. We show that the cisplatin treatment of 4T1 cells and spheroids results in a shift in metabolite levels; metabolites including nucleic acids such as DNA, 783 cm-1 with p = 0.00021 for cells; p = 0.02173 for spheroids, major amino acids such as threonine, 1338 cm-1 with p = 0.00045 for cells; p = 0.01022 for spheroids, proteins such as amide III, 1248 cm-1 with p = 0.00606 for cells; p = 0.00511 for spheroids serve as early predictors of response. Our RS findings were also applicable to canine-derived organoids, showing spatial variations in metabolic changes as a function of organoid depth in response to cisplatin. Further, the metabolic pathways such as tricarboxylic acid (TCA)/citric acid cycle and glyoxylate and dicarboxylate metabolism that drive drug response showed significant differences based on organoid depth, replicating the heterogeneous treatment response seen in solid tumors where there is a difference from the periphery to the tumor core. Our study showcases the versatility of RS as a predictive tool for treatment response applicable from cells to organotypic cultures, that has the potential to decrease animal burden and readout time for preclinical drug efficacy.

Healthcare Experiences and Health Outcomes Among Rural LGBTQ+ Individuals.

PURPOSE: To describe healthcare experiences and health outcomes among rural LGBTQ + individuals. DESIGN: 2022 cross-sectional survey. SETTING: Southern Illinois. SAMPLE: 85 individuals. MEASURES: Demographics, sexual orientation and gender identity, healthcare experiences, health outcomes. ANALYSIS: Experiences and outcomes were assessed vs orientation and identity. Distribution comparison was by t-test and chi-square, risk prediction by logistic regression, and significance assumed at P < .050. RESULTS: By orientation, participants were: 35.3% gay, 16.5% lesbian, and 45.8% bisexual plus; and by identity they were: 49.4% cisgender, 25.9% transgender, and 24.8% other identity. Survey item responses ranged from 95%-99%. Compared to gay men, lesbians and bisexual plus individuals more frequently reported medical bill payment difficulty (58.3% and 57.9% vs 25.0%; P = .020) and more past month days of poor mental health (19.4 and 15.8 vs 10.6; P = .018). Compared to heterosexual and other identity, transgender individuals less frequently reported having a routine medical provider (72.7% vs 92.7% and 95.0%; P = .037) and more frequently reported past healthcare denial (45.5% vs 17.5% and 18.8%; P = .042). Current health was associated with medical bill payment ability (OR = .33, 95% CI = .13-.86) and respectful treatment by healthcare administrators (OR = 3.90, 95% CI = 1.34-11.35) and clinicians (OR = 3.82, 95% CI = 1.39-10.47). Significance of some findings likely limited due to sample size. CONCLUSIONS: Our data describes healthcare experience and health outcome disparities among rural lesbian, gay, bisexual, transgender, queer and other sexual and gender minority individuals, and indicate that clinical experiences directly influence health outcomes.

Evaluation of different media compositions promoting hepatocyte differentiation in the canine liver organoid model.

Organoids are 3-dimensional (3D) self-assembled structures capable of replicating the microanatomy and physiology of the epithelial components of their organ of origin. Adult stem cell (ASC) derived organoids from the liver have previously been shown to differentiate into primarily mature cholangiocytes, and their partial differentiation into functional hepatocytes can be promoted using specific media compositions. While full morphological differentiation of mature hepatocytes from ASCs has not yet been reported for any species, the functional differentiation can be approximated using various media compositions. Six differentiation media formulations from published studies on hepatic organoids were used for the differentiation protocol. Target species for these protocols were humans, mice, cats, and dogs, and encompassed various combinations and concentrations of four major hepatocyte media components: Bone morphogenetic protein 7 (BMP7), Fibroblast Growth Factor 19 (FGF19), Dexamethasone (Dex), and Gamma-Secretase Inhibitor IX (DAPT). Additionally, removing R-spondin from basic organoid media has previously been shown to drive the differentiation of ASC into mature hepatocytes. Differentiation media (N = 20) were designed to encompass combinations of the four major hepatocyte media components. The preferred differentiation of ASC-derived organoids from liver tissue into mature hepatocytes over cholangiocytes was confirmed by albumin production in the culture supernatant. Out of the twenty media compositions tested, six media resulted in the production of the highest amounts of albumin in the supernatant of the organoids. The cell lines cultured using these six media were further characterized via histological staining, transmission electron microscopy, RNA in situ hybridization, analysis of gene expression patterns, immunofluorescence, and label-free proteomics. The results indicate that preferential hepatocyte maturation from canine ADC-derived organoids from liver tissue is mainly driven by Dexamethasone and DAPT components. FGF19 did not enhance organoid differentiation but improved cell culture survival. Furthermore, we confirm that removing R-spondin from the media is crucial for establishing mature hepatic organoid cultures.

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.

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.

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.

Molecular signatures of retinal ganglion cells revealed through single cell profiling.

Retinal ganglion cells can be classified into more than 40 distinct subtypes, whether by functional classification or transcriptomics. The examination of these subtypes in relation to their physiology, projection patterns, and circuitry would be greatly facilitated through the identification of specific molecular identifiers for the generation of transgenic mice. Advances in single cell transcriptomic profiling have enabled the identification of molecular signatures for cellular subtypes that are only rarely found. Therefore, we used single cell profiling combined with hierarchical clustering and correlate analyses to identify genes expressed in distinct populations of Parvalbumin-expressing cells and functionally classified RGCs. RGCs were manually isolated based either upon fluorescence or physiological distinction through cell-attached recordings. Microarray hybridization and RNA-Sequencing were employed for the characterization of transcriptomes and in situ hybridization was utilized to further characterize gene candidate expression. Gene candidates were identified based upon cluster correlation, as well as expression specificity within physiologically distinct classes of RGCs. Further, we identified Prph, Ctxn3, and Prkcq as potential candidates for ipRGC classification in the murine retina. The use of these genes, or one of the other newly identified subset markers, for the generation of a transgenic mouse would enable future studies of RGC-subtype specific function, wiring, and projection.

FGF23 Induction of O-Linked N-Acetylglucosamine Regulates IL-6 Secretion in Human Bronchial Epithelial Cells.

The hexosamine biosynthetic pathway (HBP) generates the substrate for the O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of proteins. The HBP also serves as a stress sensor and has been reported to be involved with nuclear factor of activated T-cells (NFAT) activation, which can contribute to multiple cellular processes including cell metabolism, proliferation, and inflammation. In our previously published report, Fibroblast Growth Factor (FGF) 23, an important endocrine pro-inflammatory mediator, was shown to activate the FGFR4/phospholipase Cγ (PLCγ)/nuclear factor of activated T-cells (NFAT) signaling in chronic inflammatory airway diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Here, we demonstrate that FGF23 increased the O-GlcNAc modification of proteins in HBECs. Furthermore, the increase in O-GlcNAc levels by FGF23 stimulation resulted in the downstream activation of NFAT and secretion of interleukin-6 (IL-6). Conversely, inhibition of FGF23 signaling and/or O-GlcNAc transferase (OGT)/O-GlcNAc reversed these effects. Collectively, these data suggest that FGF23 induced IL-6 upregulation and secretion is, at least, partially mediated via the activation of the HBP and O-GlcNAc levels in HBECs. These findings identify a novel link whereby FGF23 and the augmentation of O-GlcNAc levels regulate airway inflammation through NFAT activation and IL-6 upregulation in HBECs. The crosstalk between these signaling pathways may contribute to the pathogenesis of chronic inflammatory airway diseases such as COPD and CF as well as metabolic syndromes, including diabetes.