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.

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.

Aspiration, but not injection, decreases cultured equine mesenchymal stromal cell viability.

BACKGROUND: Recently, equine multipotent mesenchymal stromal cells (MSC) have received significant attention as therapy for various conditions due to their proposed regenerative and immune-modulating capacity. MSC are commonly administered to the patient through a hypodermic needle. Currently, little information is available on the effect of such injection has on equine MSC immediate and delayed viability. We hypothesize that viability of equine MSC is not correlated with needle diameter during aspiration and injection. RESULTS: Using a 3 mL syringe, manual injection of equine cord blood (CB) or bone marrow-derived (BM) MSC with no needle and needles ranging in size from 18 to 30 Ga did not affect immediate MSC viability. Similarly, 24 h post-injection, MSC delayed viability was not different between any of the tested needles as determined by a resazurin-based proliferation assay. Using a 3 mL syringe, aspiration of MSC through 20, 25, and 30 Ga needles resulted in significant decreases in immediate viability with no change in delayed viability when compared to aspiration without a needle. BM- and CB-MSC were observed to be of similar size with a diameter ± SD of 19.8 ± 2.7 and 20.4 ± 2.2 µm, respectively. In comparison, the smallest needles, (30 Ga) have an internal diameter of 160 µm. CONCLUSIONS: Following injection, needle diameter did not affect immediate or delayed viability of equine MSC. Following aspiration through needles sizes 20 Ga and smaller, immediate viability, but not delayed viability, decreased. As a result, an 18 Ga or larger needle should be utilized for aspiration of cell suspensions. In contrast, needle selection for MSC injection should be based on clinical preference and experience rather than concerns over decreasing MSC viability.

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.

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.

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.

Characterization and Immunomodulatory Effects of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

BACKGROUND: Mesenchymal stromal cells (MSC) hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies. HYPOTHESIS AND OBJECTIVES: We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC (derived from the same dogs) will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1) proliferation rate, 2) cell surface marker expression, 3) DNA methylation levels, 4) potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5) immunomodulatory potency in vitro. RESULTS: 1) AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days) for passage (P) 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21). 2) Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3) Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4) Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-ß3)-based induction medium. 5) Immunomodulatory capacity was equal regardless of cell source when tested in mitogen-stimulated lymphocyte reactions. Priming of MSC with pro-inflammatory factors interferon-gamma and/or tumour necrosis factor did not increase the lymphocyte suppressive properties of the MSC compared to untreated MSC. CONCLUSIONS/SIGNIFICANCE: No significant differences were found between AT- and BM-MSC with regard to their immunophenotype, progenitor, and non-progenitor functions. Both MSC populations showed strong adipogenic and osteogenic potential and poor chondrogenic potential. Both significantly suppressed stimulated peripheral blood mononuclear cells. The most significant differences found were the higher isolation success and proliferation rate of AT-MSC, which could be realized as notable benefits of their use over BM-MSC.

Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

BACKGROUND: Mesenchymal stromal cells (MSC) are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS) is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL) has proven to be a suitable alternative to FBS for expansion of human MSC. HYPOTHESIS AND OBJECTIVES: We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1) isolation, 2) proliferation, 3) spontaneous differentiation, and 4) directed differentiation. RESULTS: 1) Medium with 10% PL was unable to isolate MSC. 2) MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT) or 30% (BM) enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3) Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4) MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis. CONCLUSIONS/SIGNIFICANCE: Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects.