Equine Regenerative Medicine Research Articles

Characterization of senescent mesenchymal stem/stromal cells derived from equine bone marrow and the effects of NANOG on the senescent phenotypes.

In equine regenerative medicine using bone marrow-derived mesenchymal stem/stromal cells (BM-MSC), the importance of the quality management of BM-MSC has been widely recognized. However, there is little information concerning the relationship between cellular senescence and the stemness in equine BM-MSC. In this study, we showed that stemness markers (NANOG, OCT4, SOX2 and telomerase reverse transcriptase) and colony forming unit-fibroblast apparently decreased accompanied with incidence of senescence-associated β-galactosidase-positive cells by repeated passage. Additionally, we suggested that down-regulation of cell proliferation in senescent BM-MSC was related to increased expression of cyclin-dependent kinase inhibitor 2B (CDKN2B). On the other hand, forced expression of NANOG into senescent BM-MSC brought upregulation of several stemness markers and downregulation of CKDN2B accompanied with restoration of proliferation potential and osteogenic ability. These results suggested that expression of NANOG was important for the maintenance of the stemness in equine BM-MSC.

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6.

Managingtendon healing process is complicated mainly due tothe limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplementedwith 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing L-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20ng/ml), (3) IM supplemented with GDF-6 (20ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combinationcould induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.

Platelet and Leukocyte Concentration in Equine Autologous Conditioned Plasma Are Inversely Distributed by Layer and Are Not Affected by Centrifugation Rate

Background: Platelet rich plasma (PRP) is used extensively in equine regenerative medicine. Differences in preparation protocols give rise to significant variability in the cellular composition of PRP making it very difficult to establish a standard of care in the field. This study aimed to optimize the preparation protocol for leukocyte-reduced PRP (P-PRP).Methods: Blood (100 mL) was collected from horses (n = 5) and divided into 2 purple top EDTA tubes and 6 (15 mL) double syringesa with a final concentration of 10% acid citrate dextrose anticoagulant. Six double syringesa were collected from each horse; PRP samples were prepared in duplicate and centrifuged at 1,100 rpm (188 × g), 1,300 rpm (263 × g), or 1,500 rpm (350 × g). Duplicates were subjected to +/– braking at the end of centrifugation. The total volume of PRP generated was measured and divided into thirds. Each third (top, middle, and bottom) were drawn off separately using the inner (6 mL syringe) and placed in purple top EDTA tubes. Automated complete blood counts were performed on all peripheral whole blood and PRP samples.Results: The concentration of leukocytes was higher in the bottom layer of PRP compared to the top and middle layers (p < 0.0001). The concentration of platelets was slightly lower in the bottom layer of PRP than the middle layer (p = 0.02). Centrifugation braking increased the leukocyte concentration in the top (p = 0.03) and middle layers of PRP (p = 0.001). Centrifugation rate had no effect on the cellular composition of PRP (p = 0.1–0.6).Conclusions: Because layer of plasma affected both platelet and leukocyte concentrations in PRP, the most important modification for the current single spin, double syringe, plasma based PRP preparation protocols is to exclude the bottom 1/3 layer of PRP.

Therapeutic mesenchymal stromal stem cells: Isolation, characterization and role in equine regenerative medicine and metabolic disorders.

Mesenchymal stromal cells (MSC) have become a popular treatment modality in equine orthopaedics. Regenerative therapies are especially interesting for pathologies like complicated tendinopathies of the distal limb, osteoarthritis, osteochondritis dissecans (OCD) and more recently metabolic disorders. Main sources for MSC harvesting in the horse are bone marrow, adipose tissue and umbilical cord blood. While the acquisition of umbilical cord blood is fairly easy and non-invasive, extraction of bone marrow and adipose tissue requires more invasive techniques. Characterization of the stem cells as a result of any isolation method, is also a crucial step for the confirmation of the cells' stemness properties; thus, three main characteristics must be fulfilled by these cells, namely: adherence, expression of a series of well-defined differentiation clusters as well as pluripotency. EVs, resulting from the paracrine action of MSCs, also play a key role in the therapeutic mechanisms mediated by stem cells; MSC-EVs are thus largely implicated in the regulation of proliferation, maturation, polarization and migration of various target cells. Evidence that EVs alone represent a complex network 0involving different soluble factors and could then reflect biophysical characteristics of parent cells has fuelled the importance of developing highly specific techniques for their isolation and analysis. All these aspects related to the functional and technical understanding of MSCs will be discussed and summarized in this review.

Glycan Profiling Analysis of Equine Amniotic Progenitor Mesenchymal Cells and Their Derived Extracellular Microvesicles.

Equine amniotic mesenchymal cells (eAMCs) are involved in many mechanisms in tissue regenerative processes. Their secreted vesicles are important effectors in a wide array of biological processes, and contribute to in vivo healing of equine tendon lesions and endometrial inflammation. Glycoconjugates are involved in cellular recognition and in the efficient uptake of extracellular vesicles (EVs) by recipient cells. In this study, we evaluated the surface glycosylation pattern of eAMCs and their EVs from the eAMCs released in conditioned medium. We used a microarray procedure in which eAMCs and eAMC-EVs were spotted on microarray slides, and incubated with a panel of 14 biotinylated lectins and Cy3-conjugated streptavidin. Signal intensity was detected using a microarray scanner. Both eAMC and eAMC-EV microarrays interacted with all the lectins, indicating the presence of N- and O-linked glycans. With respect to eAMCs, eAMC-EVs, were found to be (1) enriched in Galβ1,3GalNAc terminating O-glycans, α2,3-linked sialoglycans, and high-mannose N-glycans (Con A); (2) diminished in N-acetyllactosamine, GalNAc, Gal, GlcNAc, and fucose terminating glycans; and (3) unchanged in α2,6 linked sialoglycans content. These results suggest that eAMC-EVs emerge from a specific eAMC microdomain, and that the high simultaneous presence of Galβ1,3GalNAc, α2,3 sialic acid, and high-mannose N-linked glycans may constitute markers of the eAMC-EVs. The role of these sugars in equine regenerative medicine requires further investigation.

Current status of myopathies affecting athletic horses

Muscular disorders rank among the most prevalent problems of horses competing in a broad variety of athletic disciplines, including track racing, dressage, endurance racing and Western riding disciplines. As described in this review, active scientific investigation is continuing to elucidate the different mechanisms underlying specific muscular disorders in horses, and is discovering and defining new disorders, and new methods of diagnosis, treatment and management. The flourishing field of equine rehabilitation and regenerative medicine is also driving the progressive application of a variety of modalities to the treatment and management of musculoskeletal conditions in horses. However, it is essential that this be accompanied by appropriate scientific investigation to verify the efficacy of recommended modalities and treatment protocols.

Horse hair follicles: A novel dermal stem cell source for equine regenerative medicine.

The easily accessible niche represented by skin and its appendages may serve as a promising source to complement modern regenerative medicine for horses. In humans and in animal models for human medicine, the hair follicle and its stem cell niches are well characterized. Since literature in this field of equine research is scarce, we sought to analyze cells of the dermal stem cell niche of the equine hair follicle morphologically and for a subset of markers useful for cell characterization via immunolabeling. We cultured equine forelock skin explants to obtain cultures with cells migrating from the hair follicles. Isolation of cells revealed typical fibroblast morphology with a strong tendency to aggregate and form spheroids. For immunofluorescent characterization of primary isolations, we tested an antibody panel consisting of lineage makers for the dermal compartment of the hair follicle, markers associated with an undifferentiated cell status and markers for epithelial cell types as negative controls. All antibodies used were also tested on equine skin sections. The isolated cells displayed clear profiles of dermal and undifferentiated cells. To substantiate our findings, we tested our primary isolations for established equine multipotent mesenchymal stromal cell antigen expression markers in flow cytometry experiments yielding strong convergence. The data presented here provide insights to a stem cell source in horses almost unnoticed to date. The basic investigations of the equine dermal hair follicle stem cell niche confirm the expression of standard markers used in other species and lay the foundation for future studies on this easily available adult stem cell source. © 2017 International Society for Advancement of Cytometry.

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

Corrigendum to “The Effect of PEI and PVP-Stabilized Gold Nanoparticles on Equine Platelets Activation: Potential Application in Equine Regenerative Medicine”

The aim of this work was to assess the effect of different stabilizing agents, for example, polyethylenimine (PEI) and polyvinylpyrrolidone (PVP), on gold nanoparticles (AuNPs) and their influence on equine platelet activation and release of particular growth factors. The gold nanoparticles were produced by chemical reduction of chloroauric acid. UV-Vis spectroscopy confirmed the presence of gold nanoparticles in investigated solutions. The AuNPs were incubated with whole blood at various concentrations. The morphology of platelets in PRP prepared from the blood incubated with AuNPs was characterized by scanning transmission electron microscopy, whereas the concentrations of growth factors and cytokines were evaluated by ELISA assays. The most promising results were obtained with equine platelets incubated with 5% AuNPs stabilized by PEI, which lead to secretion of bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), and fibroblast growth factor 1 (FGF-1) and simultaneously cause decrease in concentration of interleukin-1 alpha (IL-1α). The qRT-PCR confirmed ELISA test results. The incubation with 5% AuNPs stabilized by PEI leads to upregulation of BMP-2 and VEGF transcripts of mRNA level and to downregulating expression of interleukin-6 (IL-6). Obtained data shed a promising light on gold nanoparticle application for future regenerative medicine application.

The Effect of PEI and PVP-Stabilized Gold Nanoparticles on Equine Platelets Activation: Potential Application in Equine Regenerative Medicine

The aim of this work was to assess the effect of different stabilizing agents, for example, polyethylenimine (PEI) and polyvinylpyrrolidone (PVP), on gold nanoparticles (AuNPs) and their influence on equine platelet activation and release of particular growth factors. The gold nanoparticles were produced by chemical reduction of chloroauric acid. UV-Vis spectroscopy confirmed the presence of gold nanoparticles in investigated solutions. The AuNPs were incubated with whole blood at various concentrations. The morphology of platelets in PRP prepared from the blood incubated with AuNPs was characterized by scanning transmission electron microscopy, whereas the concentrations of growth factors and cytokines were evaluated by ELISA assays. The most promising results were obtained with equine platelets incubated with 5% AuNPs stabilized by PEI, which lead to secretion of bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), and fibroblast growth factor 1 (FGF-1) and simultaneously cause decrease in concentration of interleukin-1 alpha (IL-1α). The qRT-PCR confirmed ELISA test results. The incubation with 5% AuNPs stabilized by PEI leads to upregulation of BMP-2 and VEGF transcripts of mRNA level and to downregulating expression of interleukin-6 (IL-6). Obtained data shed a promising light on gold nanoparticle application for future regenerative medicine application.

Regional Limb Perfusion Of The Equine Distal Limb With Stem Cells: Lessons Learned From In Vivo Tracking Of Tc99m-HMPAO Labeled Cells

Regional Limb Perfusion Of The Equine Distal Limb With Stem Cells: Lessons Learned From In Vivo Tracking Of Tc99m-HMPAO Labeled Cells

Characterization of mesenchymal stem cells derived from the equine synovial fluid and membrane.

BackgroundIsolation of mesenchymal stem cells (MSCs) in equines, has been reported for different tissues including bone marrow, adipose, umbilical cord, peripheral blood, and yolk sac. In regard to the MSCs derived from synovial fluid (SF) or membrane (SM), there is data available for humans, dogs, pigs, goats and horses. Especially in equines, these cells have being considered promising candidates for articular regeneration. Herein, we established and characterized MSCs obtained from equine SF and SM. Samples were obtained during arthroscopy and cultured using MEM (Minimum Essential Medium). MSCs were characterized by morphology and expression of specific markers for stem cells, pluripotency, inflammation, and cell cycle.ResultsThe medium MEM was more effective (97 % ± 2) to maintain both cultures. The cultures were composed by adherent cells with fibroblast-like shape, which had a growth pattern represented by a sigmoidal curve. After the expansion, the cells were analyzed by flow cytometry for stem cells, inflammatory, and cell cycle markers, and both lineages showed significant expression of CD45, Oct3/4, Nanog, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF, and LY6a. In contrast, there were differences in the cell cycle phases between the lineages, which was not observed in relation to the mitochondrial electrical potential.ConclusionGiven the large impact that joint pathology has on the athletic performance horses, our results suggested that the SF and SM are promising sources of stem cells with satisfactory characteristics of growth and gene expression that can be used in equine regenerative medicine.

Spheroids from equine amnion mesenchymal stem cells: an in vitro study

Background : Equine amnion mesenchymal stem cells (EAMSCs) from amnion isolated after the foal birth represented an alternative source of easy collection of mesenchymal cells used in equine regenerative medicine. Methods : These cells grown as 2D culture in α-MEM medium supplemented with EGF were differentiated in adipogenic, chondrogenic and osteogenic cells. Half a million cells as pellet were left in 15ml tubes with the same differentiation media for 20 days. After the pellets were collected, embedded in paraffin for morphological study. Results : 2D culture showed EAMSCs with an embryonic phenotype (C-kit+, CD105+, Oct-4+) and a differentiation potential in adipogenic, chondrogenic and osteogenic multipotent cells. By a reproducible method of 3D culture, at day 20 the Authors evidenced a formation of small aggregated spheroids gradually gathering. In cross sections the surface of spheroid evidenced flattened cells embedded in a red matrix by Alizarin staining and occasionally a core of calcium precipitation. A network of apoptotic or necrotic cells in a not mineralized matrix was present into the center of nodules. The 3D spheroids appeared larger (mean diameter of 605±53 µm for gathering spheroids and 1486±79 µm for spheroids already gathered) than those from standard monolayer cultures (mean diameter of 200 ± 73 µm). Conclusions : EAMSCs cultured in 3D method preserve their in vitro multipotent differentiation than adherent 2D culture method. These EAMSCs included in extracellular matrix not mineralized at day 20 seem to be a good source of MSCs for tissue repair and regeneration in equine medicine.

Application of Gold Nanoparticles of Different Concentrations to Improve the Therapeutic Potential of Autologous Conditioned Serum: Potential Implications for Equine Regenerative Medicine

The aim of this work was to evaluate the effect of different concentrations of gold nanoparticles (AuNPS) on equine platelet morphology, ultrastructure as well as secretion of bone morphogenetic protein 2 (BMP‐2), vascular endothelial growth factor (VEGF), gelsolin (GSN), and tumor necrosis factor alpha (TNF‐α). The gold nanoparticles, with an average diameter of 20 nm, were incubated with whole anticoagulated blood at 1%, 5%, and 10% w/v for 24 hours. We showed that the application of gold nanoparticles at all the tested concentrations led to platelet activation; however, 5% of AuNPS resulted in the secretion of elevated levels of GSN and VEGF, while the concentration of TNF‐α was the lowest. The data indicate that this method may be considered a promising tool for improving the currently used in clinical practice commercially available autologous conditioned serum (ACS) isolation systems for the treatment of equine osteoarthritis.

State of the art: stem cells in equine regenerative medicine.

According to Greek mythology, Prometheus' liver grew back nightly after it was removed each day by an eagle as punishment for giving mankind fire. Hence, contrary to popular belief, the concept of tissue and organ regeneration is not new. In the early 20th century, cell culture and ex vivo organ preservation studies by Alexis Carrel, some with famed aviator Charles Lindbergh, established a foundation for much of modern regenerative medicine. While early beliefs and discoveries foreshadowed significant accomplishments in regenerative medicine, advances in knowledge within numerous scientific disciplines, as well as nano- and micromolecular level imaging and detection technologies, have contributed to explosive advances over the last 20 years. Virtually limitless preparations, combinations and applications of the 3 major components of regenerative medicine, namely cells, biomaterials and bioactive molecules, have created a new paradigm of future therapeutic options for most species. It is increasingly clear, however, that despite significant parallels among and within species, there is no 'one-size-fits-all' regenerative therapy. Likewise, a panacea has yet to be discovered that completely reverses the consequences of time, trauma and disease. Nonetheless, there is no question that the promise and potential of regenerative medicine have forever altered medical practices. The horse is a relative newcomer to regenerative medicine applications, yet there is already a large body of work to incorporate novel regenerative therapies into standard care. This review focuses on the current state and potential future of stem cells in equine regenerative medicine.

The current 'state of play' of regenerative medicine in horses: what the horse can tell the human.

The horse is an attractive model for many human age-related degenerative diseases of the musculoskeletal system because it is a large animal species that both ages and exercises, and develops naturally occurring injuries with many similarities to the human counterpart. It therefore represents an ideal species to use as a 'proving ground' for new therapies, most notably regenerative medicine. Regenerative techniques using cell-based therapies for the treatment of equine musculoskeletal disease have been in use for over a decade. This review article provides a summary overview of the sources, current challenges and problems surrounding the use of stem cell and non-cell-based therapy in regenerative medicine in horses and is based on presentations from a recent Havemeyer symposium on equine regenerative medicine where speakers are selected from leading authorities in both equine and human regenerative medicine fields from 10 different countries.

Epidermal-like architecture obtained from equine keratinocytes in three-dimensional cultures

Despite the high prevalence of skin conditions in the horse, there is a dearth of literature on the culture and biology of equine skin cells, and this is partially attributable to the lack of suitablein vitro skin models. The objective of this study was to develop a three-dimensional (3D) culture system that would support the proliferation and differentiation of equine keratinocytes, similar to that observed in natural epidermis. Cell monolayers were obtained from explants of equine skin and serially passaged as highly pure keratinocyte populations (> 95% of cells), based on their expression of cytokeratins, including CK-5 and CK-14, which are associated in vivo with proliferating keratinocyte populations. Explant-derived keratinocytes were seeded into Alvetex™ 3D tissue scaffolds for 30 days under conditions that promote cell differentiation. Ultrastructural, immunohistochemical and biochemical analyses revealed that keratinocytes within scaffolds were able to proliferate and attain tissue polarity, including differentiation into basal and suprabasal layers. The basal layer contained distinct cuboidal cells with large nuclei and stained for proliferative markers such as CK-5 and CK-14. In contrast, the suprabasal layers consisted of cells with distinct polyhedral morphology, abundant cytoplasmic processes and desmosomes indicative of stratum spinosum and distinct flattened cornified cells that expressed involucrin, a marker of terminal differentiation. Thus, keratinocytes derived from primary equine skin explants were able to attain epidermal-like architecture in culture. This novel system could provide a very useful tool for modelling skin diseases, drug testing/toxicity studies and, potentially, equine regenerative medicine. Copyright © 2016 John Wiley & Sons, Ltd.

Successful isolation of equine mesenchymal stromal cells from cryopreserved umbilical cord blood‐derived mononuclear cell fractions

The therapeutic potential of mesenchymal stromal cells for cellular therapy has generated increasing interest in human as well as veterinary medicine. Considerable research has been performed on the cryopreservation of expanded mesenchymal stromal cells, but little information is available on the cryopreservation of the original mononuclear cell fraction. The present study describes a protocol to expand equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood. To this end, mononuclear cells were isolated from 7 umbilical cord blood samples and cryopreserved at a concentration of 1-2 × 10(9) cells/l cold freezing solution. Cells were cryopreserved and kept frozen for at least 6 months before thawing. Frozen cryotubes were thawed in a 37°C water bath. Putative equine mesenchymal stromal cells were immunophenotyped using multicolour flow cytometry based on a selected 9 marker panel. Average cell viability upon thawing was 98.7 ± 0.6%. In 6 out of 7 samples, adherent spindle-shaped cell colonies were observed within 9.0 ± 2.6 days and attained 80% confluency at 12.3 ± 3.9 days. After 3 passages, putative equine mesenchymal stromal cells were successfully immunophenotyped as CD29, CD44 and CD90 positive, and CD45, CD73, CD79α, CD105, MHC II and monocyte-marker negative. Equine mesenchymal stromal cells can be cultured after cryopreservation of the isolated mononuclear cells, a time- as well as cost-efficient approach in equine regenerative medicine.

Een degeneratieve gewrichtsaandoening behandeld met autologe equine mesenchymale stamcellen uit het perifeer bloed

Een vijfjarige Duitse warmbloedhengst die chronisch mank was ten gevolge van een degeneratieve aandoening in het kroongewricht en die geen verbetering vertoonde na conservatieve therapieën, werd behandeld met autologe mesenchymale stamcellen (MSC). Deze MSC werden geïsoleerd uit het perifeer bloed van de patiënt en 2,5 miljoen van deze cellen werden in het gewricht geïnjecteerd, tweemaal met een interval van acht weken. De positieve evolutie na behandeling werd gedocumenteerd aan de hand van een klinische evaluatie en objectieve drukplaatanalysen. In dit artikel wordt voor de eerste maal het gebruik van perifeer bloed beschreven als bron van MSC voor het behandelen van een degeneratieve gewrichtsaandoening bij een paard.

Equine peripheral blood‐derived mesenchymal stem cells: Isolation, identification, trilineage differentiation and effect of hyperbaric oxygen treatment

Two studies report variability in proliferation and limited adipocyte differentiation of equine peripheral blood-derived adult mesenchymal stem cells, thus casting doubt on their adipogenic potential. Peripheral blood can be a valuable source of adult mesenchymal stem cells if cell culture conditions permissive for their adherence, proliferation and differentiation are defined. Hyperbaric oxygen treatment has been reported to mobilise haematopoietic progenitor stem cells into the peripheral blood in humans and mice, but similar experiments have not been done in horses. To optimise cell culture conditions for isolation, propagation and differentiation of adult stem cells from peripheral blood and to assess the effect of hyperbaric oxygen treatment on adult stem cell concentrations. Peripheral blood was collected from the jugular vein of 6 research mares, and mononuclear cells were isolated. They were subjected to cell culture conditions that promote the adherence and proliferation of adult stem cells. The cells were characterised by their adherence, expression of cellular antigen markers, and trans-differentiation. Each horse was subjected to 3 hyperbaric oxygen treatments, and stem cells were compared before and after treatments. Stem cells derived from adipose tissue were used as controls. One-third of the horses yielded viable stem cells from peripheral blood, positive for CD51, CD90 and CD105, and demonstrated osteocyte, chondrocyte and adipocyte differentiation. Hyperbaric oxygen treatment resulted in a significant increase in CD90-positive cells. Horses that did not yield any cells pretreatment did so only after 3 hyperbaric oxygen treatments. Peripheral blood can be a valuable source of adult stem cells, if one can identify reliable equine-specific markers, provide methods to increase the number of circulating progenitor cells and optimise cell culture conditions for growth and viability. Our findings are important for further studies towards technological advances in basic and clinical equine regenerative medicine.