For centuries, starting at a time when physicians cared for both human patients and their animals, human and veterinary health have been intertwined. Veterinarians, physicians, and other scientific health and environmental professionals, in an initiative now referred to as “One Health,” have started capitalizing on this approach to improve the lives of all species (1–5). Due to shared commonalities such as pathophysiology of specific disease states, co-morbidities, and extrinsic factors, which may influence treatment outcomes in people and animals, a One Health approach has the potential to better predict therapeutic success and efficiently translate promising medical advances in human and veterinary patients (2). Notably, the field of regenerative medicine can benefit from the incorporation of companion animals in the assessment of novel therapies and thereby change the trajectory of care for human and veterinary patients (4, 6–8).

The significant number of failures of phase II and III human clinical trials in reproducing the success of preclinical trials has raised awareness of model fidelity (9–11). Rodent models play an invaluable role in biomedical research; however, awareness of the beneficial role of companion animals in translational research is increasing. Naturally occurring diseases in these species caused by complex interactions between multiple genes and environmental factors may provide several distinct advantages over induced models of disease for translational studies and for discovery science for which acceptable models are lacking (9). Currently, there are at least 462 canine, 223 feline, and 132 equine potential models of human diseases associated with Mendelian traits (www.omia.org) and many more that are not associated with specific genetic causes that provide critical model features of high fidelity.

Companion animals are relatively outbred with a longer life-span and larger size permitting diagnostic and treatment options that cannot be performed in rodent models and with basic biochemical and physiological processes which more closely resemble those in humans when compared to rodents (Figure 1) (17). Imaging and longitudinal biologic sampling may not be feasible yet in rodent models, which is particularly important in monitoring for clinical efficacy and side effects associated with novel therapies to minimize veterinary and human patient risk. Furthermore, companion animals are exposed to external and environmental factors, which influence disease development, progression, impact of therapeutics, and subject these patients to traumatic injury in a manner similar to human patients (2, 5). Critical to the ongoing success and protection of these valuable models is an increased demand for sophisticated, cutting-edge care for companion animals and the resulting surge in veterinary clinical trials. These veterinary clinical trials not only provide valuable insight into efficacy and safety of therapies for extrapolation in humans but improve the standard of care for veterinary patients. Given the value of a One Health approach, we argue that regenerative therapies in veterinary species in translational and clinical practice are beneficial for the efficient advancement of the field. The goal of this commentary is to review the use of various cell types and their derived products for regenerative medicine applications in companion animals including practices from cell processing and development to distribution and administration to assist in improved translational applications.

Since every part of cell handling and processing can affect cellular phenotype, it is critical that standardized, detailed protocols are developed. Such protocols allow accurate assessment and design of comparative studies, which can help explain variable and unexpected outcomes and direct future studies, best practices, as well as scaled-up manufacturing. It is critical to acknowledge, identify, and understand cellular differences between species in order to develop species-specific cell processing protocols. Key variables include media (incl. fetal bovine serum (FBS)), oxygen, pH, cell culture substrate, cell seeding density, passaging frequency, harvesting, preservation, and distribution chain methodologies. This in turn highlights an important caveat with regards to the One Health approach in that one cell type working in one animal species may not be directly translated to a similar human type or a human condition. While all model systems have inherent limitations, the incorporation of naturally occurring disease in veterinary species along with traditional pre-clinical animal models of disease into a novel translational bio-medical research paradigm, may increase the predictive value of such data and its applicability to human medicine (44, 45).

Similar across all species and applications, effective, large-scale use of cell therapies requires quality controls similar to other drug products but with some additional challenges. Quality control must be assessed from the early stages with screening of donors, through careful monitoring during processing, and ultimately with performance, sterility, potency, and functional assays of the final, delivered product. Donor screening is dependent on species and risk assessment and can draw from experience and techniques used for safe blood transfusion and organ transplantation to decrease spread of infectious disease and to identify optimal cell donors. Specific cell therapies may require additional donor screening techniques, such as testing for chromosomal abnormalities in PSC lines, to determine maximum efficacy (52). Sterility can be assessed by direct observation of cell morphology, growth characteristics, and visual presence of infectious organisms as well as culture at various stages in product development including the final product at the time of delivery. However, challenges exist regarding the need for reliable, rapid microbial testing methods to allow safe and timely product release (53). Challenges also exist in determining the best methods for assessing cell quality, function, and potency. Validated, feasible, and clinically-relevant assays are needed and may have to be product-specific considering the variability noted in tissue type, species, and disease application (54). Continued evaluation of the target and off-target effects and mechanisms of action of individual cell therapies are needed to provide additional information to evaluate short- and long-term safety and will greatly increase the potential for cell product approval.

Cell delivery, survival, integration, and functionality are all critical in the long-term effectiveness of cellular therapies. The first decision is the administration route, which can be systemic or local. Systemic delivery has the advantage of being easier, but cells will be transported non-specifically to many areas of the body unless they are modified to home to specific locations. Alternatively, cells may be delivered directly to the desired site of action: injected into a defect (i.e., into a tendon or ligament defect where there is a core lesion and in which case there may be up to 95% cell retention) or transplanted as either a single cell suspension, as suspended cell aggregates, as micro-encapsulated clusters, or as tissue-engineered constructs. In the case of suspended single cells or aggregates, the viscosity, composition, and temperature-dependent behavior of the substrate can be manipulated to ensure fast delivery and high viability. In the case of encapsulated or tissue-engineered cell delivery, the physical, chemical, and structural properties of the biomaterial can be tailored to ensure the correct mechanical, chemical, and biological functioning of the cells/tissues. The size of companion animals allows investigation of all translationally applicable methods of cell delivery, which is not always feasible in preclinical laboratory animal models.

There has been growing interest in recipient characteristics and the ways that the variations in major histocompatibility complexes (MHC) may affect the survivability of cell products when delivered as therapies into several species (55, 56). Recent literature insinuates that repeated injections of MSCs of differing haplotypes may determine the efficacy of treatments (57). More research in this area is forthcoming.

Immune recognition and subsequent destruction of allogeneic cells administered for therapeutic purposes is a topic of great interest in the regenerative medicine field. While allogeneic MSCs were considered to be immune-privileged, numerous pre-clinical, veterinary and human clinical studies have demonstrated that while MSCs employ multiple immune-evasive mechanisms, administered MSCs induce an immune response that is, at least partially, responsible for the lack of long-term engraftment (58, 59). Specifically, several groups demonstrated the formation of alloantibody in multiple species including cats, horses, pigs, macaques, rats, and humans in response to systemic infusion of allogeneic MSCs (58, 60, 61). However, the significance of such antibody development is currently unknown with regards to clinical outcome as patients demonstrate various clinical improvement despite antibody development and repeated dosing. The innate immune system, which has a key role in the initiation of the adaptive response, is further activated by the administration of the allogeneic MSCs (62, 63). The decreased immunogenicity of allogeneic MSC is driven by multiple mechanisms. MSCs express low levels of MHC class I and no MHC class II molecules when not activated. Moreover, human MSCs also express HLA-G, a non-classical MHC molecule that suppresses effector leukocyte function and was initially described in placental trophoblasts as a key player in maternal immune tolerance (64, 65). Moreover, MSCs secrete numerous paracrine factors (e.g., IDO, NO, PGE₂, TGF-β, PD-L1 etc.) that shift classical monocytes to an immunomodulatory phenotype, suppress effector T cell activation and proliferation, and promote the differentiation of T regulatory cells.

While MSCs treatment delivers a therapeutic benefit in the absence of long-term engraftment (likely due to paracrine mechanisms), immune tolerance that enables long-term engraftment is critical for the transplantation of iPSC/EC-derived cells and tissues from mismatched donors. When human iPSCs were initially reported in 2007, hope for personal regenerative medicine application was on the horizon (26, 66). However, autologous iPSC treatments are not feasible at this time due to the high costs associated with the creation, characterization, and validation of iPSC lines from individual patients. As such, much effort is invested in efforts to create universal off-the-shelf iPSC-derived grafts. Such efforts include the creation of cryo-banks for HLA-homozygous iPSC lines to enable the clinical use of MHC-matched iPSC-derived grafts (67, 68). Efforts to create a universal iPSC line via genetic editing are further underway. Specifically, most research is targeting the deletion of MHC I to prevent cytotoxic CD8 T cell-mediated toxicity and at the same time to avoid NK attack that is driven by the lack of MHC I expression. Deletion of β2M and CIITA genes to prevent MHC-I expression and the induction of NK inhibitors such as HLA-E and CD47 have been reported (67, 69–71). Companion animals such as dogs, cats, and horses may represent a very attractive platform to study the immune compatibility of genetically modified universal iPSCs given the presence of well-defined breeds along with a more outbred population.

While numerous peer-reviewed manuscripts describing the use of MSCs in various veterinary clinical trials have been published, the Center for Veterinary Medicine (CVM) at the FDA has not yet approved any MSC or other animal cell-based therapy product for clinical use at the time of manuscript preparation (March 2021). Readers are referred to an informative recent paper reviewing veterinary clinical trials in the field of regenerative medicine (6). Currently, no central, searchable system exists for veterinary clinical trials. Animal studies are not listed on clinicaltrials.gov, and there is no requirement to publicly list animal clinical studies. It is therefore difficult to gain an overview of the field. Of note, the American Veterinary Medical Association (AVMA) operates an Animal Health Studies Database (AAHSD) that allows investigators and the general public to search for veterinary clinical trials, though trial registration is voluntary (https://ebusiness.avma.org/aahsd/study_search.aspx). A search via this database at the time of publication yielded no active studies and nine completed studies in the field of regenerative medicine. The FDA has recently launched a webpage where animal studies using cell-based products filed with FDA CVM can be listed (https://www.fda.gov/animal-veterinary/development-approval-process/clinical-field-studies-animal-cells-tissues-and-cell-and-tissue-based-products-actps). Participation is voluntary and is provided as a service by FDA to help facilitate study enrollment for clinical trials with an active FDA file that is in good standing. Other regulatory bodies are encouraged to provide similar listing services to aid in enrollment of clinical trials as well as providing an overview of the field. Active discussions focused on the development of veterinary cell-based therapy registries to track study information are ongoing.

Animal models of disease have had an undeniable contribution to human research, providing significant contributions to medical understanding and advancement and preventing potential human harm. However, preclinical animal research has an unpredictable translation to humans, which raises ethical concerns as well as represents a large use of resources with no measurable benefit. Robinson et al. noted three areas of concern with animal models of disease: study design and data analysis, inherent heterogeneity of animal and human subjects, and the translation of preclinical animal trials to human clinical trials (72). Several other papers have highlighted similar concerns including issues with induced animal disease models and concerns over the impact of captivity on study results (73). As noted above, natural animal disease models in companion animals can overcome many of these concerns, including providing beneficial treatment to the animals themselves, but one would undermine their value by assuming they are without limitations. There are generally four options for disease models available today: human subjects research, induced disease models, artificial models, and naturally occurring animal disease models. Each of these models has its strengths and weaknesses, which can vary for the disease being studied. For example, spontaneous disease models can have increased variability, require longer study time, and, in the case of regenerative medicine, there is known variability in cell function between species (74). To truly optimize resources and outcomes, veterinarians, physicians, researchers, statisticians, and regulatory agencies need to work together to define needs, characterize models, share knowledge and information, design strong and relevant studies, and correctly assess the study results. Ultimately, optimal outcomes may require combining several models in a thoughtful and coordinated fashion to create impactful, sustainable translational applications.

The development of therapeutic cell products has unique challenges that require a non-conventional, translational research approach and regulation. Specific challenges that are unique to cellular therapy include cellular engraftment, biocompatibility, and graft vs. host immune response. Moreover, given the inherent capacity of stem cells to self-renew and differentiate, stem cell-derived cellular products present unique safety challenges with delayed neoplastic transformation as a primary concern. Transplantation of human stem cell products into animal models does not model host response or graft behavior, regardless of the integrality of the recipient immune response. Given the critical significance of host-graft immune compatibility, a novel approach is warranted in which not only the disease of interest needs to be modeled but also the candidate therapy/cells.

We propose a novel paradigm for translational research of cellular therapeutic products that integrates a selective and highly informed use of spontaneous disease in animals. Veterinary clinician scientists are motivated and trained to facilitate such a paradigm shift toward a One Health approach. Academic veterinary hospitals, centers for veterinary clinical trials, and basic science laboratories are primed to provide the knowledge, infrastructure, and skill required to design and successfully execute meaningful translational research projects.

Importantly, current funding allocated by the NIH and other medical research funding agencies for such translational research projects is insufficient to capitalize on the potential benefit to human and veterinary patients. Addressing the immediate and critical need for funding and regulatory agencies to endorse companion animal, translational cellular therapies and to provide competitive monetary support for translational medicine research teams rooted within veterinary sciences would provide the means to actualize the potential of cellular therapies.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

BA: study conception and design, financial support, writing of manuscript, figure design, and final approval of manuscript. TW: study conception and design, financial support, collection of data, writing of manuscript, figure design, and final approval of manuscript. TK, SV, DB, AW, LG, MK, and AK: study conception and design, collection of data, writing of manuscript, and final approval of manuscript. All authors contributed to the article and approved the submitted version.

BA serves on the scientific advisory board of Gallant, TK serves as the founder, CEO and CSO of eQcell Inc., LG is a shareholder of Advanced Regenerative Therapies, and serve on the advisory board of eQcell Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.