Equine bone marrow-derived mesenchymal stem cells: optimization of cell density in primary culture (original) (raw)
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Veterinary Surgery, 2006
Objectives-To characterize equine bone marrow (BM)-derived mesenchymal stem cell (MSC) growth characteristics and frequency as well as their adipogenic and osteogenic differentiation potential. Study Design-In vitro experimental study. Animals-Foals (n ¼ 3, age range, 17-51 days) and young horses (n ¼ 5, age range, 9 months to 5 years). Methods-Equine MSCs were harvested and isolated from sternal BM aspirates and grown up to passage 10 to determine cell-doubling (CD) characteristics. Limit dilution assays were performed on primary and passaged MSCs to determine the frequency of colony-forming units with a fibroblastic phenotype (CFU-F), and the frequency of MSC differentiation into adipocytes (CFU-Ad) and osteoblasts (CFU-Ob). Results-Initial MSC isolates had a lag phase with a significantly longer CD time (DT ¼ 4.9 AE 1.6 days) compared with the average DT (1.4 AE 0.22 days) of subsequent MSC passages. Approximately 1 in 4224 AE 3265 of the total nucleated BM cells displayed fibroblast colony-forming activity. Primary MSCs differentiated in response to adipogenic and osteogenic inductive conditions and maintained their differentiation potential during subsequent passages. Conclusions-The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of foals and young adult horses are similar to those documented for BM MSCs of other mammalian species. Clinical Relevance-The results have direct relevance to the use of BM as a potential source of adult stem cells for tissue engineering applications in equine veterinary medicine. r
Isolation and characterization of bone marrow–derived equine mesenchymal stem cells
American Journal of Veterinary Research, 2007
Objective—To isolate and characterize bone marrow–derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. Sample Population—Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. Procedures—Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs. Results—Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The mult...
Journal of Veterinary Research, 2016
The objective of the study was immunophenotypic and cytogenetic analysis of mesenchymal stem cells from equine bone marrow and foal umbilical cords duringThe mesenchymal stem cells were obtained from equine bone marrow of three horses and from foal umbilical cords of six foals. The cells were cultured in COThe results of cytogenetic analysis of equine bone marrow mesenchymal stem cells at the third and fourth passages indicated that the level of karyotype variability of these cells corresponded to the spontaneous level of karyotype variability typical of the peripheral blood lymphocytes of this species. Equine bone marrow contained several clones of stem cells that differed in the expression of specific nuclear markers characteristic of proliferating cells.Mesenchymal stem cells from foal umbilical cords during
Stem Cells, 2006
Fibroblast-like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow-derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast-like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum-free medium in pellet cultures as a three-dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription-polymerase chain reaction evaluations. Fibroblast-like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood-derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow-derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood-derived fibroblast-like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow-derived MSCs under the conditions used in this study.
American Journal of Veterinary Research, 2009
Objective—To optimize the isolation and culture of mesenchymal stem cells (MSCs) from umbilical-cord blood (UCB), identify variables that predicted successful MSC isolation, and determine whether shipping, processing, and cryopreservation altered MSC viability, recovery rates, and expansion kinetics. Sample Population—UCB samples from 79 Thoroughbred and Quarter Horse mares. Procedures—UCB samples were processed to reduce volume and remove RBCs. Nucleated cells (NCs) were cryopreserved or grown in various culture conditions to optimize MSC monolayer expansion and proliferation. Donor and UCB-sample factors were analyzed to determine their influence on the success of MSC isolation and monolayer expansion. Results—MSCs capable of multilineage in vitro differentiation were expanded from > 80% of UCB samples. Automated UCB processing and temperature-controlled shipping facilitated sterile and standardized RBC reduction and NC enrichment from UCB samples. The number of NCs after UCB s...
American Journal of Veterinary Research, 2011
Objective—To evaluate effects of apheresis on mesenchymal stem cells (MSCs) and compare those MSCs with MSCs obtained from adipose tissue or bone marrow (BM). Sample Population—Samples obtained from 6 adult horses. Procedures—Samples of blood from a peripheral vein, adipose tissue, and BM aspirate were obtained from each horse. Samples were processed via apheresis of blood and techniques reported elsewhere for adipose tissue and BM. Cultures were maintained until adherence and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteoblastogenic, and chondrogenic potential. Results—Apheresis product had a significantly higher mononuclear percentage, higher platelet count, and lower RBC count, compared with values for peripheral blood. No cell adherence to the tissue culture plates was detected for the apheresis product. Adherence was detected for 6 of 6 adipose-derived and 4 of 6 BM-derived samples. Variations in efficiency were detected for differentiati...
Stem Cell Research & Therapy, 2014
Introduction: Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank.
Equine Bone Marrow Derived Mesenchymal Stem Cells: Isolation and Multilineage Differentiation
Objective - To evaluate growth characteristics and differentiation capacity of equine mesenchymal stem cell (eMSCs) derived from bone marrow (BM). Study design - In vitro experimental study. Animals - Four young adult horses (2-5 years old) Procedure - Cell morphology and growth characteristics of eMSCs harvested from BM were evaluated in standard culture conditions. eMSCs in passage 3 were subjected to osteogenic and adipogenic differentiation induction to investigate their differentiation potential. PCR analysis of differentiated cells was done to determine differentiation. Results - The cells expanded to sufficient quantity for therapeutic purposes within days and they survived to later passages while sustained their fibroblast-like morphology. Positive osteogenesis was detected via Alizarin Red staining and adipogenesis was confirmed by Oil Red O staining. The cells were more potential to differentiate into osteoblasts rather than adipocytes. PCR analysis approve relative expression of osteogenic and adipogenic genes. Conclusions and Clinical relevance - The results of this study further support eBM-MSCs as a cell population with the capacity to proliferate and differentiate down the osteogenic and adipogenic lineages. Equine BM is a potentials source of MSCs for cell-based regenerative therapies in horse.