One Health: Circadian Medicine Benefits Both Non-human Animals and Humans Alike.

Circadian biology's impact on human physical health and its role in disease development and progression is widely recognized. The forefront of circadian rhythm research now focuses on translational applications to clinical medicine, aiming to enhance disease diagnosis, prognosis, and treatment responses. However, the field of circadian medicine has predominantly concentrated on human healthcare, neglecting its potential for transformative applications in veterinary medicine, thereby overlooking opportunities to improve non-human animal health and welfare. This review consists of three main sections. The first section focuses on the translational potential of circadian medicine into current industry practices of agricultural animals, with a particular emphasis on horses, broiler chickens, and laying hens. The second section delves into the potential applications of circadian medicine in small animal veterinary care, primarily focusing on our companion animals, namely dogs and cats. The final section explores emerging frontiers in circadian medicine, encompassing aquaculture, veterinary hospital care, and non-human animal welfare and concludes with the integration of One Health principles. In summary, circadian medicine represents a highly promising field of medicine that holds the potential to significantly enhance the clinical care and overall health of all animals, extending its impact beyond human healthcare.

Bioprocess development for cord blood mesenchymal stromal cells on microcarriers in Vertical-Wheel bioreactors.

Equine mesenchymal stromal cells (MSCs) have been found to be beneficial for the treatment of many ailments, including orthopedic injuries, due to their superior differentiation potential and immunomodulating properties. Cell therapies require large cell numbers, which are not efficiently generated using conventional static expansion methods. Expansion of equine cord blood-derived MSCs (eCB-MSCs) in bioreactors, using microcarriers as an attachment surface, has the potential to generate large numbers of cells with increased reproducibility and homogeneity compared with static T-flask expansion. This study investigated the development of an expansion process using Vertical-Wheel (VW) bioreactors, a single-use bioreactor technology that incorporates a wheel instead of an impeller. Initially, microcarriers were screened at small scale to assess eCB-MSC attachment and growth and then in bioreactors to assess cell expansion and harvesting. The effect of different donors, serial passaging, and batch versus fed batch were all examined in 0.1 L VW bioreactors. The use of VW bioreactors with an appropriate microcarrier was shown to be able to produce cell densities of up to 1E6 cells/mL, while maintaining cell phenotype and functionality, thus demonstrating great potential for the use of these bioreactors to produce large cell numbers for cell therapies.

Overview of Equine Stem Cells: Sources, Practices, and Potential Safety Concerns.

Over the past 2 decades, equine veterinarians are turning increasingly to stem cell therapies to repair damaged tissues or to promote healing through modulation of the immune system. Research is ongoing into optimizing practices associated with stem cell product transport, dosage, and administration. Culture-expanded equine mesenchymal stem cell therapies seem safe, even when used allogeneically, but various safety concerns should be considered. Stem cells and cellular reprogramming tools hold great promise for future equine therapies.

MicroRNAs as Prognostic Markers for Chondrogenic Differentiation Potential of Equine Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are a promising cell source for cartilage tissue regeneration in animals and humans but with large interdonor variation in their in vitro chondrogenic differentiation potential. Underlying molecular mechanisms responsible for culture-expanded MSC heterogeneity remain poorly understood. In this study, we sought to identify variations in microRNA (miRNA) signatures associated with cultured equine MSC chondrogenic differentiation potential from different donors. Neocartilage tissue generated from equine cord blood-derived MSCs was categorized as having either high or low chondrogenic potential (LCP) based on their histological appearance and quantification of glycosaminoglycan deposition. Using next-generation sequencing, we identified 30 differentially expressed miRNAs among undifferentiated MSC cultures that corresponded with their chondrogenic potential. Of note, MSCs with LCP upregulated miR-146a and miR-487b-3p, which was also observed by quantitative real-time polymerase chain reaction. Our findings suggest that miRNA profiling of equine MSC cultures may have prognostic value in selecting MSC donors with regard to their chondrogenic differentiation potential.

Opening the "Black Box" Underlying Barriers to the Use of Canine Induced Pluripotent Stem Cells: A Narrative Review.

Induced pluripotent stem cells (iPSCs) are produced by resetting the epigenetic and transcriptional landscapes of somatic cells to express the endogenous pluripotency network and revert them back to an undifferentiated state. The reduced ethical concerns associated with iPSCs and their capacity for extensive self-renewal and differentiation make them an unparalleled resource for drug discovery, disease modeling, and novel therapies. Canines (c) share many human diseases and environmental exposures, making them a superior translational model for drug screening and investigating human pathologies compared to other mammals. However, well-defined protocols for legitimate ciPSC production are lacking. Problems during canine somatic cell reprogramming (SCR) yield putative ciPSCs with incomplete pluripotency, at very low efficiencies. Despite the value of ciPSCs, the molecular mechanisms underlying their unsuccessful production and how these may be addressed have not been fully elucidated. Factors, including cost, safety, and feasibility, may also limit the widespread clinical adoption of ciPSCs for treating canine disease. The purpose of this narrative review is to identify barriers to canine SCR on molecular and cellular levels, using comparative research to inform potential solutions to their use in both research and clinical contexts. Current research is opening new doors for the application of ciPSCs in regenerative medicine for the mutual benefit of veterinary and human medicine.

TRPV4 activation enhances compressive properties and glycosaminoglycan deposition of equine neocartilage sheets.

Objective: To evaluate the effect of Transient Receptor Potential Vanilloid 4 (TRPV4) cation channel modulation on mesenchymal stromal cell (MSC)-derived neocartilage. Methods: RT-PCR was performed to evaluate mRNA levels of chondrogenic, hypertrophic and candidate mechanoresponsive genes in equine neocartilage sheets exposed to pulses of the TRPV4 agonist (GSK101) at different concentrations (N â€‹= â€‹10). Biochemical assays and mechanical tests (double indentation and unconfined compression) evaluated neocartilage properties (N â€‹= â€‹5). Results: GSK101 treatment (1 â€‹nM) increased ACAN levels after treatment for 1-h per day for 3 days. No increase was detected for hypertrophic markers RUNX2, MMP13, MMP1, ALP or COL10A1 at this concentration. This treatment regimen also increased sGAG content and enhanced compressive properties compared to untreated controls. GSK101 showed no effect on candidate mechanoresponsive genes at the time-point of analysis. Conclusions: Chemical activation of TRPV4 signalling can be used as a strategy to enhance matrix synthesis and maturation of MSC-derived engineered neocartilage and augment its load-bearing capacity.

A Review of Fetal Bovine Serum in the Culture of Mesenchymal Stromal Cells and Potential Alternatives for Veterinary Medicine.

Fetal bovine serum (FBS) remains widely used as a supplement in cell culture media used in the isolation and expansion of mesenchymal stromal cells (MSC) despite longstanding practical, clinical, and ethical concerns over its use. As a result, research on alternative culture media supplement solutions that conserve crucial MSC characteristics has become increasingly relevant. Species-specific supplements and serum-free media such as platelet lysate or chemically defined media have been assessed for their effect in MSC cultures regarding proliferation, differentiation, and immunomodulatory capacity. While none of the alternatives offer a complete solution in replacing traditional FBS supplemented media for culturing MSCs for all species, short-term or transitional use of FBS-free media can perform equally well and could address some of the concerns over the use of FBS.

Corrigendum: Extracorporeal Shock Wave Therapy Enhances the In Vitro Metabolic Activity and Differentiation of Equine Umbilical Cord Blood Mesenchymal Stromal Cells.

[This corrects the article DOI: 10.3389/fvets.2020.554306.].

Steps Toward Standardized In Vitro Assessment of Immunomodulatory Equine Mesenchymal Stromal Cells Before Clinical Application.

Inflammation-associated disorders are significant causes of morbidity in horses. Equine single-donor mesenchymal stromal cells (sdMSCs) hold promise as cell-therapy candidates due to their secretory nonprogenitor functions. This has been demonstrated by mononuclear cell suppression assays (MSAs) showing that sdMSCs are blood mononuclear cell (BMC) suppressive in vitro. sdMSCs derived from umbilical cord blood are of clinical interest due to their ease of procurement, multipotency, and immunomodulatory ability. Due to the inherent donor-to-donor heterogeneity of MSCs, the development of robust and easily deployable methods of potency assessment is critical for improving MSCs' predictability in treating inflammatory diseases. This study focuses on the development of robust in vitro potency assays and the assessment of potential sdMSC therapeutic end products generated from pooled sdMSCs (pMSCs). We hypothesized that, compared to MSA using only one donor, MSA using pooled BMCs (pBMCs) is a more robust sdMSC potency assay due to reduced donor BMC heterogeneity. pBMCs were generated by pooling equine BMCs isolated from peripheral blood of five donors in equal ratios. pBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stored in liquid nitrogen until use. Similarly, pooling sdMSCs from multiple equine donors in equal ratios generated pMSCs. sdMSC cultures were assessed with pBMCs in MSA using Bromodeoxyuridine ELISA and CFSE. Proliferation assessment of BMCs from individual donors revealed varied responses to concanavalin A (ConA) stimulation. MSA using BMCs from single donors further demonstrated BMC donor variability. Utilizing this assay, we have also found that the immunosuppressive potencies of pMSCs are at least equal, if not more, than the calculated mean of individual cultures. MSA based on pBMCs provides a consistent and reproducible equine sdMSC potency assay. This knowledge could be used in production monitoring of cellular potency and as release criteria before clinical use.

Cell Therapy in Veterinary Medicine as a Proof-of-Concept for Human Therapies: Perspectives From the North American Veterinary Regenerative Medicine Association.

In the past decade, the potential to translate scientific discoveries in the area of regenerative therapeutics in veterinary species to novel, effective human therapies has gained interest from the scientific and public domains. Translational research using a One Health approach provides a fundamental link between basic biomedical research and medical clinical practice, with the goal of developing strategies for curing or preventing disease and ameliorating pain and suffering in companion animals and humans alike. Veterinary clinical trials in client-owned companion animals affected with naturally occurring, spontaneous disease can inform human clinical trials and significantly improve their outcomes. Innovative cell therapies are an area of rapid development that can benefit from non-traditional and clinically relevant animal models of disease. This manuscript outlines cell types and therapeutic applications that are currently being investigated in companion animals that are affected by naturally occurring diseases. We further discuss how such investigations impact translational efforts into the human medical field, including a critical evaluation of their benefits and shortcomings. Here, leaders in the field of veterinary regenerative medicine argue that experience gained through the use of cell therapies in companion animals with naturally occurring diseases represent a unique and under-utilized resource that could serve as a critical bridge between laboratory/preclinical models and successful human clinical trials through a One-Health approach.

Gene Expression Profile Is Different between Intact and Enzymatically Digested Equine Articular Cartilage.

OBJECTIVES: RNA isolation is necessary for the evaluation of gene expression. Due to the nature of its extracellular matrix, RNA isolation from articular hyaline cartilage is difficult and thus the tissue is commonly enzymatically digested in order to extract RNA from the obtained chondrocytes. We hypothesized that the digestion process affects the expression levels of common cartilage-associated genes. DESIGN: Expression of cartilage-associated genes was compared between intact cartilage and digested chondrocytes from weight bearing and non-weight bearing regions of the equine fetlock joint. RESULTS: The gene expression of SOX9, COL1A2, COL2A1, ACAN, and COLX were analyzed. Digested cartilage showed a significant decrease in the expression of COL1A2, COL2A1, and ACAN compared to intact cartilage in both joint regions, and an increase in COLX expression in non-weight bearing cartilage only. CONCLUSIONS: Enzymatic digestion of cartilage significantly impacts gene expression profile. We conclude that while RNA isolation from intact cartilage is more technically difficult, determination of gene expression should be conducted on intact cartilage if true representation of the in vivo processes is sought.

The use of induced pluripotent stem cells in domestic animals: a narrative review.

Induced pluripotent stem cells (iPSCs) are undifferentiated stem cells characterized by the ability to differentiate into any cell type in the body. iPSCs are a relatively new and rapidly developing technology in many fields of biology, including developmental anatomy and physiology, pathology, and toxicology. These cells have great potential in research as they are self-renewing and pluripotent with minimal ethical concerns. Protocols for their production have been developed for many domestic animal species, which have since been used to further our knowledge in the progression and treatment of diseases. This research is valuable both for veterinary medicine as well as for the prospect of translation to human medicine. Safety, cost, and feasibility are potential barriers for this technology that must be considered before widespread clinical adoption. This review will analyze the literature pertaining to iPSCs derived from various domestic species with a focus on iPSC production and characterization, applications for tissue and disease research, and applications for disease treatment.

Extracorporeal Shock Wave Therapy Enhances the In Vitro Metabolic Activity and Differentiation of Equine Umbilical Cord Blood Mesenchymal Stromal Cells.

Extracorporeal shock wave therapy (ESWT) has been shown to induce different biological effects on a variety of cells, including regulation and stimulation of their function and metabolism. ESWT can promote different biological responses such as proliferation, migration, and regenerations of cells. Recent studies have shown that mesenchymal stromal cells (MSCs) secrete factors that enhance the regeneration of tissues, stimulate proliferation and differentiation of cells, and decrease inflammatory and immune reactions. Clinically, the combination of these two therapies has been used as a treatment for tendon and ligament lesions in horses; however, there is no scientific evidence supporting this combination of therapies in vivo. Therefore, the objectives of the study were to evaluate the effects of ESWT on equine umbilical cord blood mesenchymal stromal cells (CB-MSCs) proliferative, metabolic, migrative, differentiation, and immunomodulatory properties in vitro. Three equine CB-MSC cultures from independent donors were treated using an electrohydraulic shock wave generator attached to a water bath. All experiments were performed as triplicates. Proliferation, viability, migration and immunomodulatory properties of the cells were evaluated. Equine CB-MSCs were induced to evaluate their trilineage differentiation potential. ESWT treated cells had increased metabolic activity, showed positive adipogenic, osteogenic, and chondrogenic differentiation, and showed higher potential for differentiation toward the adipogenic and osteogenic cell fates. ESWT treated cells showed similar immunomodulatory properties to none-ESWT treated cells. Equine CB-MSCs are responsive to ESWT treatment and showed increased metabolic, adipogenic and osteogenic activity, but unaltered immunosuppressive properties. In vivo studies are warranted to determine if synergistic effects occur in the treatment of musculoskeletal injuries if ESWT and equine CB-MSC therapies are combined.

Mesenchymal Stromal Cells as Potential Antimicrobial for Veterinary Use-A Comprehensive Review.

The emergence of "superbugs" resistant to antimicrobial medications threatens populations both veterinary and human. The current crisis has come about from the widespread use of the limited number of antimicrobials available in the treatment of livestock, companion animal, and human patients. A different approach must be sought to find alternatives to or enhancements of present conventional antimicrobials. Mesenchymal stromal cells (MSC) have antimicrobial properties that may help solve this problem. In the first part of the review, we explore the various mechanisms at work across species that help explain how MSCs influence microbial survival. We then discuss the findings of recent equine, canine, and bovine studies examining MSC antimicrobial properties in which MSCs are found to have significant effects on a variety of bacterial species either alone or in combination with antibiotics. Finally, information on the influence that various antimicrobials may have on MSC function is reviewed. MSCs exert their effect directly through the secretion of various bioactive factors or indirectly through the recruitment and activation of host immune cells. MSCs may soon become a valuable tool for veterinarians treating antimicrobial resistant infections. However, a great deal of work remains for the development of optimal MSC production conditions and testing for efficacy on different indications and species.

A Review of Recent Advances in 3D Bioprinting With an Eye on Future Regenerative Therapies in Veterinary Medicine.

3D bioprinting is a rapidly evolving industry that has been utilized for a variety of biomedical applications. It differs from traditional 3D printing in that it utilizes bioinks comprised of cells and other biomaterials to allow for the generation of complex functional tissues. Bioprinting involves computational modeling, bioink preparation, bioink deposition, and subsequent maturation of printed products; it is an intricate process where bioink composition, bioprinting approach, and bioprinter type must be considered during construct development. This technology has already found success in human studies, where a variety of functional tissues have been generated for both in vitro and in vivo applications. Although the main driving force behind innovation in 3D bioprinting has been utility in human medicine, recent efforts investigating its veterinary application have begun to emerge. To date, 3D bioprinting has been utilized to create bone, cardiovascular, cartilage, corneal and neural constructs in animal species. Furthermore, the use of animal-derived cells and various animal models in human research have provided additional information regarding its capacity for veterinary translation. While these studies have produced some promising results, technological limitations as well as ethical and regulatory challenges have impeded clinical acceptance. This article reviews the current understanding of 3D bioprinting technology and its recent advancements with a focus on recent successes and future translation in veterinary medicine.

The Importance of Dosing, Timing, and (in)Activation of Adipose Tissue-Derived Mesenchymal Stromal Cells on Their Immunomodulatory Effects.

Mesenchymal stromal cells (MSCs) are attractive candidates for immunomodulatory cell therapy. However, it remains unknown how far therapeutic efficacy and potency are dependent on the dosage and activity of the MSCs. We previously observed that infusion of MSCs leads to rapid and transient changes in cytokine expression in blood, lung, and liver. In the present study, increasing doses of syngeneic adipose tissue-derived MSCs were infused in healthy mice and systemic changes in G-CSF, IL6, IL-10, and CXCL5 were detected 2 h after administration of 3 × 105 MSCs per animal, but not at lower doses. In lung and liver tissue, dose-dependent effects of MSCs on cytokine mRNA expression levels were detected from doses as low as 3 × 103 MSCs. Infusion of secretome-deficient or IFNγ-activated MSCs in healthy mice had similar effects on systemic cytokine levels as control MSCs, suggesting that in vivo at least the initial systemic effect of MSC administration is independent of the level of activity of MSCs, but depends on the response of host cells to MSCs. The results of this study reveal a rapid dose-dependent effect of MSCs and stress the important role of host cells in MSC treatment. This knowledge contributes to the design of rational MSC trials and to the quest for clinical efficacy of MSC therapy.

Improved expansion of equine cord blood derived mesenchymal stromal cells by using microcarriers in stirred suspension bioreactors.

Equine mesenchymal stromal cells (MSCs) are increasingly investigated for their clinical therapeutic utility. Such cell-based treatments can require cell numbers in the millions or billions, with conventional expansion methods using static T-flasks typically inefficient in achieving these cell numbers. Equine cord blood-derived MSCs (eCB-MSCs), are promising cell candidates owing to their capacity for chondrogenic differentiation and immunomodulation. Expansion of eCB-MSCs in stirred suspension bioreactors with microcarriers as an attachment surface has the potential to generate clinically relevant numbers of cells while decreasing cost, time and labour requirements and increasing reproducibility and yield when compared to static expansion. As eCB-MSCs have not yet been expanded in stirred suspension bioreactors, a robust protocol was required to expand these cells using this method. This study outlines the development of an expansion bioprocess, detailing the inoculation phase, expansion phase, and harvesting phase, followed by phenotypic and trilineage differentiation characterization of two eCB-MSC donors. The process achieved maximum cell densities up to 75,000 cells/cm2 corresponding to 40 million cells in a 100 mL bioreactor, with a harvesting efficiency of up to 80%, corresponding to a yield of 32 million cells from a 100 mL bioreactor. When compared to cells grown in static T-flasks, bioreactor-expanded eCB-MSC cultures did not change in surface marker expression or trilineage differentiation capacity. This indicates that the bioreactor expansion process yields large quantities of eCB-MSCs with similar characteristics to conventionally grown eCB-MSCs.

Beyond Cartilage Repair: The Role of the Osteochondral Unit in Joint Health and Disease.

IMPACT STATEMENT: In this comprehensive review, we are providing a holistic overview of osteochondral tissue development, disease, pain localization, as well as structural evaluation and current repair strategies. This review is intended to serve as a broad introduction to this multidisciplinary research area. It is a thorough examination of the biological aspects of the osteochondral unit from a tissue engineering perspective, highlighting the importance of the subchondral bone in chondral and osteochondral lesion repair and pain relief.

Equine Cord Blood Mesenchymal Stromal Cells Have Greater Differentiation and Similar Immunosuppressive Potential to Cord Tissue Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are the most common cell population studied for therapeutic use in veterinary medicine. MSCs obtained from neonatal sources such as umbilical cord tissue (CT-MSCs) or cord blood (CB-MSCs) are appealing due to the non-invasive nature of procurement and the time allowed for characterization of cells before use. However, it remains unclear as to whether CB- or CT-MSCs have equivalent progenitor and non-progenitor functions. CB-MSCs have been shown to have superior chondrogenic potential to MSCs from other sources, whereas their immunomodulatory capacity does not seem to vary significantly. Using equine CB-MSCs and CT-MSCs from the same donors, we hypothesized that MSCs from both sources would have a similar immunophenotype, that CB-MSCs would be more amenable to differentiation, and that they can equally suppress lymphocyte proliferation. We evaluated cells from both sources for "classic" equine MSC markers CD90, CD105, CD29, and CD44, as well as pericyte markers CD146, NG2, and α-SMA. Contrary to our hypothesis, CB-MSCs showed mid- to high expression of pericyte surface markers CD146 and NG2, whereas expression in CT-MSCs was absent. On trilineage differentiation, CB-MSCs were more osteogenic and chondrogenic based on alkaline phosphatase activity and glycosaminoglycan content, respectively. Finally, using a mononuclear cell (MNC) suppression assay, we determined that both CB-MSCs and CT-MSCs are capable of suppressing stimulated MNC proliferation to a similar degree. We have determined that the choice of MSC tissue source should be made with the intended application in mind. This appears to be particularly relevant if pursuing a progenitor-based treatment strategy.

Cell Identity, Proliferation, and Cytogenetic Assessment of Equine Umbilical Cord Blood Mesenchymal Stromal Cells.

The aim of the present work was to determine proliferation capacity, immunophenotype and genome integrity of mesenchymal stromal cells (MSCs) from horse umbilical cord blood (UCB) at passage stage 5 and 10. Passage 4 cryopreserved UCB-MSCs from six unrelated donors were evaluated. Immunophenotypic analysis of UCB-MSC revealed a cell identity consistent with equine MSC phenotype by high expression of CD90, CD44, CD29, and very low expression of CD4, CD11a/18, CD73, and MHC class I and II antigens. Proliferative differences were noted among the UCB-MSC cultures. UCB-MSCs karyotype characteristics at passage 5 (eg, 2n = 64; XY, or XX) included 20% polyploidy and 62% aneuploidy. At passage 10, the proportion of polyploidy and aneuploidy was 21% and 82%, respectively, with the increase in aneuploidy being significant compared with passage 5. Furthermore, conventional GTG-banded karyotyping revealed several structural chromosome abnormalities at both passage 5 and 10. The clinical relevance of such chromosome instability is unknown, but determination of MSC cytogenetic status and monitoring of patient response to MSC therapies would help address this question.