Optimisation of bone marrow aspiration from the equine sternum for the safe recovery of mesenchymal stem cells

Reasons for performing study: Mesenchymal stem cell (MSC) therapy for orthopaedic disease is being used with increasing frequency; there is a need to define a safe, reliable and effective technique for the recovery of MSCs from the sternum of the horse. Objectives: To describe an optimised safe technique for obtaining bone marrow‐derived MSCs from the sternum of the Thoroughbred horse. Methods: The anatomical relationship of the sternum with the heart and internal anatomy was demonstrated in cadavers. Sternal anatomy was evaluated ultrasonographically and after midline sectioning. Sternebrae were examined histologically after aspiration to determine the effect of needle insertion. The quality of the aspirate was evaluated as the number of colony‐forming units from sequential and separately aspirated 5 ml aliquots and assessed for their multipotency using trilineage differentiation. Results: The optimal safe location for the needle was the 5th sternebra because it had a safe dorsoventral thickness and was cranial to the apex of the heart. This sternebra could be reliably identified ultrasonographically. Aspirates could also be obtained from the 4th and 6th sternebrae, although the former is between the front limbs and the latter closer to the heart. Minimal disruption of the internal bony architecture was seen after needle insertion through the thin outer cortex and the first 5 ml aliquot contained the greatest number of colony‐forming units of mesenchymal stem cells with trilineage capabilities. Conclusions: Accurate placement of a Jamshidi needle into the medullary cavity of the 4th–6th individual sternebrae is facilitated by the use of ultrasonography and enables aspiration of bone marrow reliably with minimal damage to the sternum and risk to the horse. Potential clinical relevance: Sternal marrow aspiration as described is a safe and reliable technique to obtain MSCs for orthopaedic cell‐based therapies.     

Isolation,growth and differentiation of equine mesenchymal stem cells: effect of donor,source, amount of tissue and supplementation with basic fibroblast growth factor

Mesenchymal stem cells (MSC) are increasingly used as therapeutical aid for the orthopaedic injuries in the horse. MSC populate different tissues but the most commonly used for clinical purposes are isolated from bone marrow or adipose tissue. The first objective of this study was to investigate if the donor animal, the tissue of origin and the technique of isolation could influence the number of MSC available for transplantation after a short-term expansion. The second aim was to devise a culture system capable of increasing MSC lifespan and we tested the effect of basic fibroblast growth factor (bFGF). Results indicate that MSC can be efficiently isolated from both sources and supplementation of bFGF enhances proliferation rate maintaining differentiation potential. In addition, this study shows that collection, expansion and storage of frozen MSC can be performed for later therapeutic use.     

Mesenchymal stem cells and bone regeneration

OBJECTIVE: To review the role of mesenchymal stem cells (MSC) in bone formation and regeneration, and outline the development of strategies that use MSC in bone healing and regeneration. STUDY DESIGN: Literature review. METHODS: Medline review, synopses of authors' published research. RESULTS: The MSC is the basic cellular unit of embryologic bone formation. Secondary bone healing mimics bone formation with proliferation of MSC then their differentiation into components of fracture callus. Bone regeneration, where large amounts of bone must form, mimics bone healing and can be achieved with MSC combined with strategies of osteogenesis, osteoinduction, osteoconduction, and osteopromotion. MSC based strategies first employed isolated and culture expanded stem cells in an osteoconductive carrier to successfully regenerate a critical segmental defect in the femur of dogs, which was as effective as autogenous cancellous bone. Because MSC appeared to be immunologically privileged, a study using mismatched allogeneic stem cells demonstrated that these cells would regenerate bone without inciting an immunologic response, documenting the possibility of banked allogeneic MSC for bone regeneration. A technique was developed for selectively retaining MSC from large bone marrow aspirates at surgery for bone regeneration. These techniques utilized osteoconductive and osteoinductive carriers and resulted in bone regeneration that was similar to autogenous cancellous bone. CONCLUSION: MSC can be manipulated and combined with carriers that will result in bone regeneration of critically sized bone defects. CLINICAL RELEVANCE: These techniques can be employed clinically to regenerate bone and serve as an alternative to autogenous cancellous bone.     

Enhanced tyrosine hydroxylase expression in PC12 cells co-cultured with feline mesenchymal stem cells

Mesenchymal stem cells (MSCs) secrete a variety of neuroregulatory molecules, such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor, which upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells. Enhancing TH gene expression is a critical step for treatment of Parkinson''s disease (PD). The objective of this study was to assess the effects of co-culturing PC12 cells with MSCs from feline bone marrow on TH protein expression. We divided the study into three groups: an MSC group, a PC12 cell group, and the combined MSC + PC12 cell group (the co-culture group). All cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum for three days. Thereafter, the cells were examined using western blot analysis and immunocytochemistry. In western blots, the co-culture group demonstrated a stronger signal at 60 kDa than the PC12 cell group (p<0.001). TH was not expressed in the MSC group, either in western blot or immunocytochemistry. Thus, the MSCs of feline bone marrow can up-regulate TH expression in PC12 cells. This implies a new role for MSCs in the neurodegenerative disease process.     

Subconjunctival bone marrow‐derived mesenchymal stem cell therapy as a novel treatment alternative for equine immune‐mediated keratitis: A case series

Equine immune‐mediated keratitis (IMMK) leads to increased corneal opacity and inflammation secondary to an alteration of the local immune system. Bone marrow‐derived mesenchymal stem cells (BM‐MSC) have been shown to modulate the immune system by downregulating inflammation. Four horses with unilateral IMMK poorly responsive to traditional medical treatments underwent novel, autologous subconjunctival BM‐MSC therapy. Bone marrow was harvested and processed as previously described for equine orthopedic disease. Horses received autologous subconjunctival BM‐MSC injections approximately every 3‐4 weeks for 1‐5 treatments total. Horses were maintained on their current medical treatment regimen throughout the BM‐MSC treatment period. Three horses had a positive response to therapy as demonstrated by an increase in corneal clarity, a decrease in neovascularization and a reduction in surface irregularity. One horse was nonresponsive to therapy. These experimental results demonstrate the safety and potential efficacy of an innovative solution for IMMK.     

Pancytopenia Secondary to Lymphoid Leukemia in Three Horses

Pancytopenia was observed in two 3-year-old geldings and one 11-year-old mare. All horses had a brief history (2 days to 4 weeks) of fever, anorexia, and depression. One of the three horses had blast cells present on a peripheral blood smear. Examination of the bone marrow showed substantial infiltration with neoplastic lymphoid cells. At necropsy, neoplastic cells were restricted to the bone marrow in one horse, present in bone marrow, liver, and spleen in the second horse, and reported in multiple tissues in the third horse, including bone marrow, kidneys, lung, myocardium and lymph nodes. The value of a bone marrow aspirate and core biopsy in the investigation of pancytopenia is highlighted. (Journal of Veterinary Internal Medicine 1993; 7:360–363. Copyright © 1993 by the American College of Veterinary Internal Medicine.)     

The canine epiphyseal-derived mesenchymal stem cells are comparable to bone marrow derived-mesenchymal stem cells

Mesenchymal stem cells (MSCs) hold great potential in cell therapy and have attracted increasing interests in a wide range of biomedical sciences. However, the scarcity of MSCs and the prolonged isolation procedure limited the clinical application. To address these 2 issues, we developed a method to isolate MSCs from bone biopsy tissues of euthanized canine body donors. Compared to the traditional method to isolate MSCs from aspirated bone marrow (BMSCs), the isolation procedure for MSCs from harvested epiphyseal cancellous bone (EMSCs) was less time-consuming. The isolated EMSCs had similar plastic-adherence, tri-lineage differentiation and consistent surface marker profiles compared to BMSCs. We harvested BMSCs and EMSCs from 24 euthanized cases from clinics and 42 euthanized donors from a local shelter. The successful rate for EMSC isolation is significantly higher compared to BMSC isolation, while the other properties of the isolated MSCs including the clonogenicity, proliferative potentials and molecular phenotypes were not discernibly different between the MSCs established by the two methods. In conclusion, we demonstrated a new procedure to harvest MSCs by bone biopsy at the epiphyseal region. This method is less time consuming and more reliable, and the resulting MSCs are comparable to those harvested by bone marrow aspiration. The combination of the two methods can greatly improve the efficiency to harvest MSCs.     

Bone marrow necrosis and myelophthisis: manifestations of T‐cell lymphoma in a horse

Abstract: A 14‐year‐old spayed American Paint mare was evaluated for mild colic, anorexia, pyrexia, and pancytopenia. Physical examination revealed mild tachycardia, tachypnea, and pale mucous membranes. Serial laboratory analyses revealed progressive pancytopenia, hyperfibrinogenemia, and hyperglobulinemia. A few large atypical cells were observed in peripheral blood smears. Results of tests for equine infectious anemia and antipenicillin antibody were negative. Serum protein electrophoresis indicated a polyclonal gammopathy. Smears of bone marrow aspirates contained hypercellular particles, but cell lines could not be identified because the cells were karyolytic, with pale basophilic smudged nuclei and lack of cellular detail. A diagnosis of bone marrow necrosis was made. Treatment consisted of antimicrobials, nonsteroidal anti‐inflammatory drugs, and corticosteroids. The pyrexia resolved; however, the pancytopenia progressively worsened and petechiation and epistaxis developed. The horse was humanely euthanized. Postmortem examination revealed a diffuse round cell neoplasm infiltrating the kidneys, spleen, lymph nodes, lungs, and bone marrow. Immunophenotyping results (CD3+, CD79α−) indicated the neoplastic cells were of T‐cell lineage. Infiltration of lymphoma cells into the bone marrow appeared to have resulted in severe myelophthisis and bone marrow necrosis. Bone marrow necrosis has been associated previously with lymphoma in humans and dogs. To our knowledge, this is the first reported case of lymphoma resulting in bone marrow necrosis in a horse.     

Characterization of Nucleated Cells From Equine Adipose Tissue and Bone Marrow Aspirate Processed for Point-of-Care Use

The objective of this study was to compare nucleated cell fractions and mesenchymal stromal cells (MSCs) from adipose tissue to bone marrow processed by a point-of-care device that are available for immediate implantation. A paired comparison using adipose and bone marrow from five horses was done. The number of nucleated cells, viability, total adherent cells on day 6 of culture and colony-forming unit fibroblasts (CFU-Fs) were determined. Gene expression for markers of stemness, adipogenic, chondrogenic, osteogenic lineage, and collagen formation was measured in total RNA isolated from adherent adipose and bone marrow cells. Day 6 adherent adipose-derived MSC was frozen briefly, whereas day 6 adherent bone marrow–derived MSC was passaged two additional times to obtain adequate cell numbers for chondrogenic, osteogenic, and adipogenic cell differentiation assays. The total cell count per gram was significantly greater for bone marrow, whereas total adherent cells per gram and the CFU-F per million nucleated cells on day 6 were significantly greater for the adipose. In undifferentiated adherent cells, relative gene expression for CD34, adipogenic, and chondrogenic markers and collagen II was significantly lower in the adipose-derived cells. Conversely, expression of collagen I was significantly higher in the undifferentiated adipose-derived cells. Cell density and total RNA were higher in differentiated adipogenic and osteogenic cultures of adipose cells and in chondrogenic cultures of bone marrow cells. This cell preparation method provides a stromal vascular fraction with a large proportion of multipotent MSCs. There are differences in the cells obtained from the two sources. This method can provide an adequate number of multipotent cells from adipose tissue for immediate implantation.     

Comparison of bone marrow aspiration at the sternum and the tuber coxae in middle-aged horses

The objective of this study was to compare bone marrow (BM) aspirates from the sternum and the tuber coxae of middle-aged horses. Bone marrow was obtained from the sternum and both tubera coxae of 12 healthy, 13-year-old geldings. Two different puncture techniques were used for the tuber coxae. The 2 syringes used for sternal sampling were evaluated separately. The mononuclear cell (MNC) fraction of the BM was isolated and the mesenchymal stem cells (MSCs) were culture-expanded. At the sternum, BM aspiration was always possible. Bone marrow aspiration at the tuber coxae required straight and deep needle penetration combined with high negative pressure. With this technique a median sample amount of 11.0 mL with large individual variation was obtained. A median of 3.06 × 10(6) MNC/mL BM (1st syringe) and 2.46 × 10(6) MNC/mL BM (2nd syringe) was isolated from sternal samples. In contrast, the tuber coxae yielded a median of 0.27 × 10(6) MNC/mL BM. The first passage yielded a median of 2.19 × 10(6) MSC (1st syringe) and 1.13 × 10(6) MSC (2nd syringe) from sternal samples, compared to a significantly lower median number of MSC from tuber coxae BM (0.06 × 10(6) MSC). The number of MNC and MSC obtainable from the BM aspirates taken from the tuber coxae is significantly lower than that obtained from the sternal BM aspirates. Autologous BM for the equine athlete is particularly clinically relevant at an advanced age. Based on our findings, the tuber coxae cannot be recommended for BM aspiration in middle-aged horses.     

Treatment of Bilateral Medial Femoral Condyle Articular Cartilage Fissures in a Horse Using Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells

The objective of this study was to describe the use, and outcome, of multipotent mesenchymal stromal cells (MSCs) in the treatment of equine articular cartilage defects of the medial femoral condyle. A 4-year-old Thoroughbred gelding (n = 1) with bilateral stifle athroscopy was found to have bilateral articular cartilage fissure defects of the medial femoral condyles with concurrent cranial cruciate ligament injury. Bone marrow derived MSCs were isolated, expanded, and suspended in a partially autologous fibrin glue. The initial cell/fibrin glue mixture was delivered arthroscopically into the articular cartilage defects 90 days after the initial arthroscopic examination. Follow-up treatments included two additional injections of MSCs suspended in lactated Ringers solution, 5 and 13 months after the initial examination, directly into the joint. Post-treatment outcome was assessed by arthroscopic examination and by comparison of preinjury and post-treatment performance records. Arthroscopic evaluation 4 months after the initial MSC treatment revealed marked smoothing, reduction in the depth of cartilage defects and observation of moderate improvement in the cranial cruciate ligament. Approximately 15 months after the initial MSC treatment the horse returned to racing. Analysis of race records demonstrated that the post-treatment (including all three MSC treatments) average race earnings (earnings per start) were comparable with those predating the initial injury. The favorable clinical response in the face of an unknown, but likely, guarded prognosis suggest that MSC therapy is not deleterious and may augment healing of articular cartilage fissures of the medial femoral condyle. MSCs represent a viable and promising alternative therapy in the treatment of articular cartilage injuries in performance horses.     

Immunoglobulin A monoclonal gammopathy in two horses with multiple myeloma

The clinical findings in two horses with secretory multiple myeloma and secondary immunoglobulin A (IgA) monoclonal gammopathy were non-specific and included weight loss, pale mucous membranes, limb oedema and bacterial respiratory tract infection. Consistent laboratory abnormalities included hyperproteinaemia, hyperglobulinaemia, hypoalbuminaemia and hypercalcaemia. The diagnosis was based on the presence of IgA monoclonal gammopathy in serum and urine and bone marrow plasmacytosis (> 10 per cent). One horse was euthanased; it had neoplastic plasma cell infiltrates in its kidneys, spleen, liver, bone marrow, myocardium and adrenal glands. The other horse was treated for a bacterial pneumonia and was still alive six months after it was first examined.     

Long-term bone marrow culture systems: normal and cyclic hematopoietic dogs.

A continuous long-term liquid culture in both a micro and macro system that incorporates bone marrow cells from normal and cyclic hematopoietic dogs is described. An adherent layer composed of fibroblasts, endothelial cells, mononuclear phagocytic cells, and fat-containing cells is essential for continuous hematopoiesis. Hematopoiesis was measured by the recovery of the nonadherent cells and the generation of committed granulocyte-monocyte progenitor cells for a period of seven weeks. Optimum growth factors include the use of horse serum, fetal bovine serum, dog serum, hydrocortisone, a 33 degrees C incubation temperature and feeding twice a week. As is true for both human and murine marrow liquid cultures, horse serum and hydrocortisone are essential for development and maintenance of fat-containing cells in the described systems. Both factors are important in hematopoiesis but their respective roles have not been defined. Normal and cyclic hematopoietic dogs bone marrow cells are comparable in their ability to establish long-term cultures. The micro-method (Linbro-well culture) gave similar results in maintaining hematopoiesis as did a macromethod (flask culture).     

Effect of Biodegradable Gelatin β-Tri Calcium Phosphate Sponges Containing Mesenchymal Stem Cells and Bone Morphogenetic Protein-2 on Equine Bone Defect

This study evaluated the osteoanagenetic effects of administering biodegradable gelatin β-tri calcium phosphate sponges containing mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) on equine bone defect. Six healthy Thoroughbred horses were used in this study. Horses were anesthetized, and skin incisions were made on all the limbs. Splint bones were exposed and a 1-cm bone defect was created in each exposed bone. Gelatin β-tri calcium phosphate sponges containing MSC and BMP-2 (MSC + BMP-2 sponge), MSC only (MSC sponge), BMP-2 only (BMP-2 sponge), or saline (saline sponge) were implanted into each bone defect at random. Defects were monitored for 16 weeks by radiography followed by computed tomography (CT) and histologic analyses. At 16 weeks, radiographic scores of MSC + BMP-2 sponge-treated defects were significantly higher than those of saline-treated defects (P = .027). Moreover, the CT value of the MSC + BMP-2 sponge group was significantly higher than that of the other groups (P = .027; P = .046; and P = .027, respectively), and the histologic score of the MSC + BMP-2 sponge group was significantly greater than that of the saline sponge group (P = .041). We conclude that MSC + BMP-2 sponge administration to bone defects accelerates bone regeneration in equines.     

Immunohistochemical localization of alpha 2-beta 1-glycoprotein in horses

Alpha 2-beta 1-glycoprotein may be found free in horse serum or complexed with alpha-1-proteinase inhibitor to form pre-alpha 2-elastase inhibitor. There has been little information published concerning alpha 2-beta 1-glycoprotein and its possible tissue sources in horses. A peroxidase-antiperoxidase technique was used to identify alpha 2-beta 1-glycoprotein in buffy coat and bone marrow neutrophils of healthy horses. Macrophages and neutrophils in bronchoalveolar lavage samples from clinically normal horses and from horses with chronic pulmonary disease also were positive for alpha 2-beta 1-glycoprotein. Alpha 2-beta 1-glycoprotein was identified in some instances in normal equine hepatocytes of formalin-fixed liver sections. In formalin-fixed lung sections from horses with chronic, small-airway disease and chronic bronchointerstitial pneumonia, alpha 2-beta 1-glycoprotein was observed in some airway secretions and in macrophages.     

Subleukaemic acute myeloid leukaemia with myelodysplasia in a horse

Acute myeloid leukaemia (AML) is rarely reported in horses and myelodysplastic syndrome (MDS) has been described only in one case. Acute myeloid leukaemia is defined as the presence of at least 20% blasts in the marrow or blood. On the other hand, MDS is characterised by morphologic abnormalities in one or more cell lineages with hypercellular marrow and peripheral cytopenias due to ineffective haematopoiesis. We report a case of acute myelomonocytic leukaemia with myelodysplasia-related features in a horse. A supposed diagnosis was based on abnormal morphology of circulating neoplastic cells and bone marrow cytology. A final diagnosis was made by using flow cytometry (FC) in conjunction with cytochemistry (CC) rarely reported in the haematopoietic neoplasms of the horse.     

Thermostable antigens from the bone marrow of several animal species.

Studies on bone marrow extracts from ten animal species have revealed the presence of thermostable antigens. Similar antigens were found in other organs as shown by immunodiffusion and cross-reactivity was demonstrated between the thermostable proteins from horse and dog and between cow and sheep proteins. Using an immunoenzymatic reaction with glucose oxidase as the marker, the thermostable antigens were localized in the blood and bone marrow polymorphonuclear leukocytes and the myeloid cell line.     

In vitro comparison of feline bone marrow-derived and adipose tissue-derived mesenchymal stem cells

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for a variety of different diseases in human and veterinary medicine. At present, MSC are most often collected from bone marrow (BM) or adipose tissue (AT) and enriched and expanded in vitro before being transferred into recipients. However, little is known regarding the culture characteristics of feline BM-derived (BM-MSC) versus AT-derived MSC (AT-MSC). We compared BM-MSC and AT-MSC from healthy cats with respect to in vitro growth and cell surface phenotype. Mesenchymal stem cells isolated from AT proliferated significantly faster than BM-MSC. Phenotypic differences between BM-MSC and AT-MSC were not present in the surface markers assessed. We conclude that BM-MSC and AT-MSC are similar phenotypically but that cultures of AT-MSC are easier to generate because of their higher intrinsic proliferative rate. Thus, AT-MSC may be the preferred MSC for clinical applications where rapid and efficient generation of MSC is important.