Equine Umbilical Cord Research Articles

Equine platelet lysate as an alternative to fetal bovine serum in equine mesenchymal stromal cell culture - too much of a good thing?

Multipotent mesenchymal stromal cells (MSC) are often culture-expanded invitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Randomised dose escalation study. Platelet concentrate was generated from 5 equine whole blood samples through a double centrifugation method and standardised to 1×10(12) platelets/l prior to a freeze/thaw cycle to produce PL. Pooled PL or pooled FBS was added to EM at concentrations of 5% to 60%. Proliferation of 4 equine CB-MSC cultures was determined after 4days using a resazurin semiquantitative assay. Cord blood-MSC proliferated with a dose-dependent response with no significant difference found between PL and FBS up to a 30% concentration. Beyond 30%, proliferation fell in the PL-cultured cells, while continued dose-dependent proliferation was noted in the FBS-cultured cells. Despite reduced cell numbers in high PL concentrations, live/dead staining revealed that adherent cells remained viable. Expansion medium enriched with PL can support short-term equine CB-MSC proliferation at conventional culture concentrations. Based on the unexpected suppression of CB-MSC at higher PL concentrations, an invivo dose study is indicated to investigate if combinational therapies of CB-MSC and platelet-rich plasma are associated with synergistic or antagonistic effect on CB-MSC function.

Phenotypical and functional characteristics of mesenchymal stem cells derived from equine umbilical cord blood.

Mesenchymal stem cells (MSCs) offer promise as therapeutic aid in the repair of tendon and ligament injuries in race horses. Fetal adnexa is considered as an ideal source of MSCs due to many advantages, including non-invasive nature of isolation procedures and availability of large tissue mass for harvesting the cells. However, MSCs isolated from equine fetal adnexa have not been fully characterized due to lack of species-specific markers. Therefore, this study was carried out to isolate MSCs from equine umbilical cord blood (UCB) and characterize them using cross-reactive markers. The plastic-adherent cells could be isolated from 13 out of 20 (65%) UCB samples. The UCB derived cells proliferated till passage 20 with average cell doubling time of 46.40±2.86h. These cells expressed mesenchymal surface markers but did not express haematopoietic/leucocytic markers by RT-PCR and immunocytochemistry. The phenotypic expression of CD29, CD44, CD73 and CD90 was shown by 96.36±1.28, 93.40±0.70, 73.23±1.29 and 46.75±3.95% cells, respectively in flow cytometry, whereas, reactivity against the haematopoietic antigens CD34 and CD45 was observed only in 2.4±0.20 and 0.1±0.0% of cells, respectively. Osteogenic and chondrogenic differentiation could be achieved using established methods, whereas the optimum adipogenic differentiation was achieved after supplementing media with 15% rabbit serum and 20ng/ml of recombinant human insulin. In this study, we optimized methodology for isolation, cultural characterization, differentiation and immunophenotyping of MSCs from equine UCB. Protocols and markers used in this study can be employed for unequivocal characterization of equine MSCs.

Evaluation of ultrasmall superparamagnetic iron oxide contrast agent labeling of equine cord blood and bone marrow mesenchymal stromal cells.

To evaluate the efficacy and effects of labeling equine umbilical cord blood (UCB)- and bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) with an ultrasmall superparamagnetic iron oxide (SPIO) contrast agent and the detection of labeled MSCs by use of MRI. UCB MSCs from placental tissues of 5 foals and BM MSCs from 5 horses. UCB and BM MSC cultures were seeded in duplicate (5,000 cells/cm(2)). One duplicate was incubated with SPIO (50 μg/mL); the other was processed identically, but without SPIO. Mesenchymal stromal cells were expanded in triplicates for 5 passages and assessed for viability and proliferative capacity, labeling efficacy, and labeled cell proportion. For MRI detection, 5 × 10(6) labeled BM MSCs from passage 1 or 2 were injected into a collagenase-induced superficial digital flexor tendon defect of an equine cadaveric forelimb from 2 horses. For passages 1, 2, and 3, labeling efficacy and cell proportion for UCB MSCs (99.6% [range, 98.8% to 99.9%], 16.6% [range, 6.5% to 36.1%], and 1.0% [range, 0.4% to 2.8%], respectively) were significantly higher than for BM MSCs (99.2% [range, 97.8% to 99.7%], 4.5% [range, 1.6% to 11.8%], and 0.2% [range, 0.1% to 0.6%], respectively). Labeling was not detectable after passage 3. Viability of MSCs was not affected, but cell doubling time increased in labeled MSCs, compared with that of unlabeled MSCs. On MRI 3-D T2*-weighted fast gradient echo sequences, decreased signal intensity was observed for BM passage 1 MSCs. Equine UCB and BM MSCs were labeled with SPIO at high efficiencies.

Intra-articular administration of xenogeneic neonatal Mesenchymal Stromal Cells early after meniscal injury down-regulates metalloproteinase gene expression in synovium and prevents cartilage degradation in a rabbit model of osteoarthritis

The anti-inflammatory and anti-catabolic effects of neonatal Mesenchymal Stromal Cell (MSC) were investigated in a xenogeneic model of mild osteoarthritis (OA). The paracrine properties of MSC on synoviocytes were further investigated invitro. OA was induced by medial meniscal release (MMR) in 30 rabbit knees. A single early (day 3) or delayed (day 15) intra-articular (IA) injection of MSC isolated from equine Umbilical Cord Wharton's jelly (UC-MSC) was performed. Rabbits were euthanized on days 15 or 56. OA grading was performed and gene expression of inflammatory cytokines and metalloproteinases was measured in synovial tissue. Paracrine effects of UC-MSC were investigated using UC-conditioned vs control medium on rabbit primary synoviocytes stimulated with interleukin 1 beta invitro. No adverse local or systemic responses were observed clinically after xenogeneic UC-MSC injection. At study end point, cartilage fibrillation was lower in early treatment than in delayed treatment group. Cellular infiltrate was observed in the synovium of both UC-MSC groups. OA synovium exhibited a reduced expression of metalloproteinases-1, -3, -13 in the early cell-treated group at d56. Invitro, UC-conditioned medium exerted anti-inflammatory and anti-catabolic effects on synoviocytes exposed to pro-inflammatory stimulus. Early IA injection of equine UC-MSC was effective in preventing OA signs in rabbit knees following MMR. UC-MSC target the synovium and modulate the gene expression pattern of synoviocytes to promote an anti-catabolic environment. This confirms the synovium is a major target and mediator of MSC therapy, modulating the expression of matrix-degrading enzymes.

Absence of maternal cell contamination in mesenchymal stromal cell cultures derived from equine umbilical cord tissue

This study aimed to determine whether maternal cell contamination exists in cells derived from equine umbilical cord tissue, a perspective material for cell-based therapies in veterinary medicine. Potential maternal cell contamination was analyzed at DNA level via a set of 16 microsatellite markers in cells originating from the cord tissue of 22 foals. In these cells no maternal cell contamination was detected at a sensitivity level of 0.01%. Our results suggest that equine umbilical cord tissue-derived cells are entirely of fetal origin.

Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood

Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73 proteins, but tested negative for CD34 and CD45. In flow cytometry, CD29, CD44, CD73, and CD90 were expressed by 96.36 ± 1.28%, 93.40 ± 0.70%, 73.23 ± 1.29% and 46.75 ± 3.95% cells, respectively. The UCB-MSCs could be differentiated to tenocytes by culturing in growth medium supplemented with 50 ng/ml of BMP-12 by day 10. The differentiated cells showed the expression of mohawk homeobox (Mkx), collagen type I alpha 1 (Col1α1), scleraxis (Scx), tenomodulin (Tnmd) and decorin (Dcn) by RT-PCR. In addition, flow cytometry detected tenomodulin and decorin protein in 95.65 ± 2.15% and 96.30 ± 1.00% of differentiated cells in comparison to 11.30 ± 0.10% and 19.45 ± 0.55% cells, respect vely in undifferentiated control cells. The findings support the observation that these cells may be suitable for therapeutic applications, including ruptured tendons in racehorses.

197 INDUCED PLURIPOTENT STEM CELLS (iPS) DERIVED FROM EQUINE UMBILICAL CORD CELLS USING LENTIVIRUS VECTOR Stemcca

Research on induced pluripotent stem cells (iPS) emerged to overcome the limitations of embryonic stem cells, such as ethical issues, security, compatibility, and availability. The nuclear reprogramming induced by viral vectors aims to induce differentiated cells to an embryonic pluripotent state. The iPS cells can be generated using retroviral vector expressing Oct4, Sox2, Klf4 and c-Myc, but produces much genomic integration (GI) which limit its use for therapeutic purpose. Alternatively, lentiviral vectors have been used to be safe and equally effective in producing iPS. Despite several cell types can be reprogramed, there is no information of which is the best cell type to be used in the generation of iPS. The umbilical cord is a reserve of multipotent mesenchymal stem cells and may present a greater reprogramming efficiency compared with fibroblasts in the generation of iPS. Here we describe the use of a single lentiviral vector composed by the combination of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) for the generation of iPS cells using equine umbilical cord (UC) cells. Therefore, samples were collected from 5 equine UC at birth. The umbilical matrices were subjected to enzymatic digestion in a solution of 0.004% collagenase diluted in PBS, and the cells obtained by filtration were plated in plastic culture bottles with 5 mL of DMEM supplemented with 20% fetal calf serum, antibiotics, and antimycotics, followed by incubation at 37°C in a 100% humid atmosphere at 5% CO2 in air. When the cells reached 40% of confluence and a concentration of 105 cells, these cells were transduced with 50 μL Human Stemcca cre-excisable constitutive polycistronic (oskm) lentivirus (EMD Millipore Corp., Billerica, MA, USA) produced according manufacturer's protocol plus 8 ng mL–1 polybrene (hexadimethrine bromide, Sigma, St. Louis, MO, USA). The culture medium was renewed 12 h after incubation. Five days after transduction, cells were transferred to murine embryonic fibroblasts (MEF) feeder layer and cultured for 14 days in a specific medium for iPS. The morphologically similar colonies to the embryonic stem cells were visualised after two weeks of infection. When the clones were well established two mechanical and two enzymatic passages were performed. Cells were re-expanded under new MEFs and submitted to alkaline phosphatase activity detection (Leukocyte Alkaline Phosphatase Kit, Sigma) according to manufacturer's recommendations. Briefly, cell cultures were fixed, incubated with a mixture of alkaline naphthol AS-BI with fast red violet LB. Red labelling insoluble deposits indicated the sites of alkaline phosphatase activity. In all cultures tested (n = 10) the expression of alkaline phosphatase was detected. The cell culture samples will still be tested for gene expression of pluripotency factors. The combination of all factors in a single transcript was efficient for reprogramming cells from the umbilical cord and allowed the derivation of mesenchymal cells in equine iPS. The use of a single lentiviral reprogramming vector represents a powerful tool for the study of iPS technology and its possible therapeutic application.

Hematological and biochemical profile of equine umbilical cord blood and comparison with peripheral blood of mare and newborn foal

Hematological and biochemical profile of equine umbilical cord blood and comparison with peripheral blood of mare and newborn foal

Characterization and clinical application of mesenchymal stem cells from equine umbilical cord blood

Tendinitis of the superficial digital flexor tendon (SDFT) is a significant cause of lameness in horses; however, recent studies have shown that stem cells could be useful in veterinary regenerative medicine. Therefore, we isolated and characterized equine umbilical cord blood mesenchymal stem cells (eUCB-MSCs) from equine umbilical cord blood obtained from thoroughbred mares during the foaling period. Horses that had tendinitis of the SDFT were treated with eUCB-MSCs to confirm the therapeutic effect. After eUCB-MSCs transplantation, the core lesion in the SDFT was found to decrease. These results suggest that transplantation using eUCB-MSCs could be another source of cell treatment.

Refinement of Culture Conditions for Maintenance of Undifferentiated Equine Umbilical Cord Blood Stem Cells

Umbilical cord blood (UCB) was collected from Thoroughbred mares at foaling, and stem cells were isolated. UCB stem cells were cultured in growth media (GM), GM + Fibroblast Growth Factor 2, GM + Flt3/Thrombopoietin/c-Kit ligand, or conditioned media. Population doubling times (PDTs) were calculated. UCB stem cells cultured in standard GM exhibited faster PDTs (30.07 ± 1.59 hours) than those cultured in any treatment media. PDT was further accelerated by cultivation in GM on fibronectin (26.99 ± 1.57 hours)-, collagen I (26.21 ± 1.18 hours)-, or gelatin (27.32 ± 1.50 hours)-coated surfaces. Time in culture increased PDT regardless of media or substrata conditions. Because Oct4, nanog, KLF4, Sox2, and c-myc are implicated in embryonic stem cell pluripotency, total RNA was harvested from UCB stem cells as well as mouse ES cells and analyzed by reverse-transcription polymerase chain reaction. Both populations expressed all transcripts. The persistence of Oct4, nanog, and Sox2 expression in UCB stem cells was monitored over time in culture. Oct4 was detected throughout the duration of the experiment. Sox2 and nanog expression was lost with time in culture. Cells cultured on protein matrices maintained nanog expression longer than those on uncoated plasticware. These phenotypes remain unaffected by the type of culture matrix and FGF2. However, the plasticity markers are lost with serial passage.

In Vitro Studies of Horse Umbilical Cord Matrix-Derived Cells: From Characterization to Labeling for Magnetic Resonance Imaging

Despite the increasing use of cell-based therapies for equine orthopedic problems, many questions remain to be answered, such as what is: the optimal cell source for particular injuries, the best timing and route of treatment and the long-term safety and efficacy of the procedure? Previously, equine mesenchymal stem cells (MSCs) have most frequently been isolated from bone marrow (BM) and adipose tissue. However, these cells have limited potential in terms of in vitro proliferation ability and differentiation capacity with increasing donor age and in vitro passage number. In addition, procurement of BM-derived cells in horses requires an invasive BM aspiration procedure which has been associated with pneumopericardium. Fetal adnexa could provide a useful alternative source of MSCs avoiding these limitations. To investigate this, we isolated and characterized presumptive stem cells from the intervascular and perivascular portions of equine umbilical cord matrix using enzymatic digestion. The cells isolated from the intervascular portion showed faster doubling times than cells from the perivascular portion (which are probably more highly differentiated). Cells from both portions expressed MSC mRNA markers (CD29, CD105, CD44, CD166) and were negative for CD34 and MHC-II. Osteogenic, adipogenic, chondrogenic and neurogenic differentiation were confirmed by specific staining and gene expression. To investigate the potential of umbilical cord-derived cells for use in cell therapies, pre-clinical experiments involving labeling of cells with magnetic resonance contrast agents (superparamagnetic iron oxide particles - SPIO - and manganese chloride) and the subsequent in vitro study of these were conducted. The SPIO labeling procedure proved to be an efficient and non toxic tool that merits further investigation and the possible development of in vivo studies.

The effects of freeze/thawing process on cryopreserved equine umbilical cord blood-derived mesenchymal stem cells

The use of mesenchymal stem cells (MSCs)-derived equine umbilical cord blood in cell grafts transplantation is advantageous; therefore, preservation of these cells is of utmost importance in repair therapies. To evaluate the viability ratio of the MSCs obtained from equine umbilical cord blood after cryopreservation, umbilical cord blood obtained from nine 46–48-week-old foals were purified for harvesting MCs. The purified cells were frozen from the first to tenth passages and stored in liquid nitrogen. After thawing, the cell viability was assessed through trypan blue staining procedure. The highest viability (>80%) ratio was observed with the cells derived from the first passage in 1 and 8 weeks after cryopreservation. However, the viability of cells was dependent on the passage used for cryopreservation. Results in this study demonstrated that equine umbilical cord blood stem cells could successfully be frozen and stored in liquid nitrogen for 8 weeks without significant change in the characterization of the cells cryopreserved as regards their viability, growth ability, and differentiation potential.

Optimization of the Isolation, Culture, and Characterization of Equine Umbilical Cord Blood Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) represent a promising population for supporting new clinical concepts in cellular therapy. A wide diversity of isolation procedures for MSC from umbilical cord blood (UCB) has been described for humans. In contrast, a few data are available in horses. In the current study, a sedimentation method using hydroxyethyl starch and a method based on the lysis of red blood cells using ammonium chloride (NH(4)Cl) were compared with two density gradient separation methods (Ficoll-Paque and Percoll). Adherent cell colonies could be established using all four isolation methods. The mononuclear cell recovery after Percoll separation, however, resulted in significantly more putative MSC colonies; and, therefore, this isolation method was used for all further experiments. Culture conditions such as cell density and medium or serum coating of the wells did not significantly affect putative MSC recovery. Isolated MSC using Percoll were subsequently differentiated toward the osteogenic, chondrogenic, and adipogenic lineage. In addition, MSC were phenotyped by multicolor flow cytometry based on their expression of different cell protein markers. Cultured MSC were CD29, CD44, and CD90-positive and CD79α, Macrophage/Monocyte and MHC II-negative. In conclusion, this study reports optimized protocols to isolate, culture, and characterize solid equine MSC from UCB.

Size-sieved subpopulations of mesenchymal stem cells from intervascular and perivascular equine umbilical cord matrix

Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites. Umbilical cords were processed for retrieval of MSCs. Unsieved cells from intervascular and perivascular portions were evaluated for cell cycle analysis and for immunophenotyping by flow cytometry. Cells from each site were separated into larger and smaller sieved populations using multi-dishes with 8-μm pore transwell inserts. Each cell population was characterized in terms of renewal capability, specific marker expression and differentiation potential. Cryopreservation was performed on sieved cells only. Cells from both areas expressed MSC and pluripotential specific markers and were able to differentiate into mesodermic and ectodermic lineages. The sieving procedure yielded two relatively homogeneous subpopulations with comparable characteristics. Surprisingly, after sieving, large intervascular and small perivascular cells were the most rapidly replicating cells [20.53 and 19.49 cell population doublings (PD) after 31 days respectively] and also showed higher fibroblast colony forming unit frequency. Unsieved cell populations were used as controls, and showed PD of 9.42(intervascular cells) and 8.54 (perivascular cells) after 31 days. Here, cells from UCM represented an intermediate stage between pluripotent embryonic and adult stem cells. Size-sieving can be used to isolate more rapidly proliferating cell populations.

Osteogenic differentiation of equine cord blood multipotent mesenchymal stromal cells within coralline hydroxyapatite scaffolds in vitro

To investigate the osteogenic differentiation potential of equine umbilical cord blood-derived multipotent mesenchymal stromal cells (CB-MSC) within coralline hydroxyapatite scaffolds cultured in osteogenic induction culture medium. Scaffolds seeded with equine CB-MSC were cultured in cell expansion culture medium (control) or osteogenic induction medium (treatment). Cell viability and distribution were confirmed by the MTT cell viability assay and DAPI nuclear fluorescence staining, respectively. Osteogenic differentiation was evaluated after 10 days using reverse transcription polymerase chain reaction, alkaline phosphatase activity, and secreted osteocalcin concentration. Cell morphology and matrix deposition were assessed by scanning electron microscopy (SEM) after 14 days in culture. Cells showed viability and adequate distribution within the scaffold. Successful osteogenic differentiation within the scaffolds was demonstrated by the increased expression of osteogenic markers such as Runx2, osteopontin, osteonectin, collagen IA; increased levels of alkaline phosphatase activity; increased osteocalcin protein secretion and bone-like matrix presence in the scaffold pores upon SEM evaluation. These results demonstrate that equine CB-MSC maintain viability and exhibit osteogenic potential in coralline hydroxyapatite scaffolds when induced in vitro . Equine CB-MSC scaffold constructs deserve further investigation for their potential role as biologically active fillers to enhance bone-gap repair in the horse.

307 INVOLVEMENT OF THE CALCIUM SENSING RECEPTOR IN GROWTH AND PROLIFERATION OF STEM CELLS FROM EQUINE UMBILICAL CORD MATRIX

The calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in extracellular Ca2+ concentration ([Ca2+]o), and external Ca2+ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca2+]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca2+]o (0.37 mM), (B) high [Ca2+]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca2+]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca2+]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the >8-μm cell line, the addition of NPS R-467, in the presence of [Ca2+]o, significantly increased cell growth after day 7 of culture (C v. A and B; P < 0.001). Increasing [Ca2+]o was not effective in this cell line (B v. A; not significant). In the <8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P < 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca2+]o increase (B v. A; P < 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P < 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes. Financial support was provided by Fondi di Ateneo 2009; University of Bari Aldo Moro (COD. ORBA09UDWX) (Resp. Sci. Maria Elena Dell’Aquila).

Three types of anomalous vasculature in the equine umbilical cord

Summary Anomalies in the number of major blood vessels in the umbilical cord are well documented in man and have been associated with both placental abnormalities and congenital problems in the neonate. This paper describes 5 cases of anomalies in the number of umbilical arteries or veins within the equine umbilical cord at term. In 4 cases, the anomalies affected vessels in the amniotic part of the cord, and in one case, vessels in the allantoic part. In all the cases placental morphology was essentially normal and fetal health and post natal development were not compromised as a result of the unusual arrangement of blood vessels within the cord.

Optimization of Culture Conditions for Equine Umbilical Cord Blood Derived Stem Cells

Optimization of Culture Conditions for Equine Umbilical Cord Blood Derived Stem Cells

Equine umbilical cord blood contains a population of stem cells that express Oct4 and differentiate into mesodermal and endodermal cell types

Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon, ligament, and bone damage suffered by sport horses. The objective of the study was to identify and characterize stem-like cells from newborn foal umbilical cord blood (UCB). UCB was collected and MSC isolated using human reagents. The cells exhibit a fibroblast-like morphology and express the stem cell markers Oct4, SSEA-1, Tra1-60 and Tra1-81. Culture of the cells in tissue-specific differentiation media leads to the formation of cell types characteristic of mesodermal and endodermal origins. Chondrogenic differentiation reveals proteoglycan and glycosaminoglycan synthesis as measured histochemically and Sox9 and collagen 2A1 gene transcription. Osteocytes capable of mineral deposition, osteonectin and Runx2 transcription were evident. Hepatogenic cells formed from UCBs express albumin and cytokeratin 18. Multinucleated myofibers that express desmin were observed indicating partial differentiation into mature muscle cells. Interestingly, conventional human protocols for UCB differentiation into adipocytes were unsuccessful in foal UCB and adult horse adipose-derived MSC. These results demonstrate that equine UCB can be induced to form multiple cell types that underlie their value for regenerative medicine in injured horses. In addition, this work suggests that subtle differences exist between equine and human UCB stem cells.

Characterization and differentiation of equine umbilical cord-derived matrix cells

Stem cells are being evaluated in numerous human clinical trials and are commercially used in veterinary medicine to treat horses and dogs. Stem cell differentiation, homing to disease sites, growth and cytokine factor modulation, and low antigenicity contribute to their therapeutic success. Bone marrow and adipose tissue are the two most common sources of adult-derived stem cells in animals. We report on the existence of an alternative source of primitive, multipotent stem cells from the equine umbilical cord cellular matrix (Wharton’s jelly). Equine umbilical cord matrix (EUCM) cells can be cultured, cryogenically preserved, and differentiated into osteo-, adipo-, chondrogenic, and neuronal cell lineages. These results identify a source of stem cells that can be non-invasively collected at birth and stored for future use in that horse or used as donor cells for treating unrelated horses.