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1.
Objective— To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs). Study Design— In vitro experimental study. Animals— Adult Thoroughbred horses (n=11). Methods— BM (5 horses; mean [±SD] age, 4±1.4 years) or adipose tissue (6 horses; mean age, 3.5±1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis±transforming growth factor‐3 (TGFβ3) and bone morphogenic factor‐6 (BMP‐6). Pellets harvested after 3, 7, 14, and 21 days were examined for cross‐sectional size and tissue composition (hematoxylin and eosin), glycosaminoglycan (GAG) staining (Alcian blue), collagen type II immunohistochemistry, and by transmission electron microscopy. Pellet GAG and total DNA content were measured using dimethylmethylene blue and Hoechst DNA assays. Results— Collagen type II synthesis was predominantly observed in MSC pellets from Day 7 onward. Unlike ASC cultures, MSC pellets had hyaline‐like matrix by Day 14. GAG deposition occurred earlier in MSC cultures compared with ASC cultures and growth factors enhanced both MSC GAG concentrations (P<.0001) and MSC pellet size (P<.004) after 2 weeks in culture. Conclusion— Equine MSCs have superior chondrogenic potential compared with ASCs and the equine ASC growth factor response suggests possible differences compared with other species. Clinical Relevance— Elucidation of equine ASC and MSC receptor profiles will enhance the use of these cells in regenerative cartilage repair.  相似文献   

2.
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 multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicate that bone marrow-derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.  相似文献   

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Since the discovery of bone marrow derived stromal cell osteogenesis in the 1960s, tissue engineering with adult multipotent stromal cells (MSCs) has evolved as a promising approach to restore structure and function of bone compromised by injury or disease. To date, accelerated bone formation with MSCs has been demonstrated with a variety of tissue engineering strategies. Though MSC bone tissue engineering has advanced over the last few decades, limitations to clinical translation remain. A current review of this promising field is presented with a specific focus on equine investigations.  相似文献   

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6.
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=3, age range, 17-51 days) and young horses (n=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=4.9+/-1.6 days) compared with the average DT (1.4+/-0.22 days) of subsequent MSC passages. Approximately 1 in 4224+/-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.  相似文献   

7.
Reason for performing the study: There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Materials and methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteristics (self‐renewal (CFU) and multilineage differentiation capacity) were determined for all 3 protocols. Results: The mean ± s.d. MSC yield from the Percoll protocol was significantly higher (6.8 ± 3.8%) than the Classic protocol (1.3 ± 0.7%). The numbers of MSCs recovered after 14 days culture per 10 ml BM sample were 24.0 ± 12.1, 14.6 ± 9.5 and 4.1 ± 2.5 × 10 6 for the Percoll, Ficoll and Classic protocols, respectively, significantly higher for the Percoll compared with the Classic protocol. Importantly, no significant difference in cell viability or in osteogenic or chondrogenic differentiation was identified between the protocols. At Passage 0, cells retrieved with the Ficoll protocol had lower self‐renewal capacity when compared with the Classic protocol but there was no significant difference between protocols at Passage 1. There were no significant differences between the 3 protocols for the global frequencies of CFUs at Passage 0 or 1. Conclusions and clinical relevance: These data suggest that the Percoll gradient density separation protocol was the best in terms of MSC yield and self‐renewal potential of the MSCs retrieved and that MSCs retrieved with the Ficoll protocol had the lowest self‐renewal but only at passage 0. Then, the 3 protocols were equivalent. However, the Percoll protocol should be considered for equine MSC isolation to minimise culture time.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

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11.
Adult Mesenchymal Stem Cells (MSC) are cells that can be defined as multipotent cells able to differentiate into diverse lineages, under appropriate conditions. These cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Initially discovered in bone marrow, MSC can now be isolated from a wide spectrum of adult and foetal tissues. Studies to evaluate the therapeutic potential of these cells are based on their ability to arrive to damaged tissues. In this paper we have done a comparative study analyzing proliferation, surface markers and OCT4, SOX9, RUNX2, PPARG genes expression in MSC cells from Bone marrow (BMMSC) and Adipose tissue (ASC). We also analyzed the role of Stem Cell Factor (SCF) on MSC proliferation and on ASCs metalloproteinases MMP-2, MMP-9 secretion. Healthy dogs were used as BMMSC donors, and ASC were collected from omentum during elective ovariohysterectomy surgery. Both cell types were cultured in IMDM medium with or without SCF, 10% Dog Serum (DS), and incubated at 38 °C with 5% CO2. Growth of BMMSCs and ASCs was exponential until 25–30 days. Flow citometry of MSCs revealed positive results for CD90 and negative for CD34, CD45 and MCH-II. Genes were evaluated by RT-PCR and metalloproteinases by zymografy. Our findings indicate morphological and immunological similarities as well as expression of genes from both origins on analyzed cells. Furthermore, SCF did not affect proliferation of MSCs, however it up-regulated MMP-2 and MMP-9 secretion in ASCs. These results suggest that metalloproteinases are possibly essential molecules pivoting migration.  相似文献   

12.
In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT‐PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue‐derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue‐derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue‐derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow‐derived MSC in dog.  相似文献   

13.
The exposure effect of cryoprotectant agents (CPAs) on morphology of preantral follicles (PAFs), stromal cell and PAF densities, and area of equine ovarian fragments were evaluated. Three independent experiments with identical methodologies were performed. Each experiment was composed of one CPA (dimethyl sulfoxide, ethylene glycol, or propylene glycol) and was performed in three replicates. Ovarian biopsy fragments were harvested from six mares in each experiment and submitted to the cryoprotectants using four times of exposure (0, 10, 15, and 20 minutes). PAF and stromal cell densities, and area of the fragments were not affected (P > .05) by any of the CPAs throughout the time of exposure. However, the morphology of the PAFs was affected (P < .05) by the CPAs. In the propylene glycol and dimethyl sulfoxide, higher (P < .05) percentages of abnormal PAFs were observed at 10 and 20 minutes of exposure, respectively. The PAF morphology in the ethylene glycol treatments was not affected (P > .05) throughout the times of exposure. Positive correlations (r = 0.57–0.77; P < .001, power = 96%–99%) were identified between PAF density and stromal cell density in all experiments. In conclusion, (1) ethylene glycol seems to be a less harmful CPA to equine PAFs, (2) exposure to CPAs did not affect the cell density and area of ovarian fragments, (3) PAF density was positively correlated with stromal cell density, and (4) stromal cell density did not affect the morphology of PAFs.  相似文献   

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15.
Equine bone marrow–derived mesenchymal stem cells (BM-eMSCs) have been used in veterinary clinics and research worldwide. For clinical use, in vitro propagation of BM-eMSCs is required (at least 2 weeks) to yield sufficient cell number for transplantation. Many culture media have been used to propagate human and animal MSCs but there are very few comparative studies of equine mesenchymal stem cells (eMSCs). The best culture media must promote cell proliferation and maintain eMSC properties. The objective of this study was to compare five basic commercial culture media by examining the proliferation rate and a representative MSC gene expression profile. Five culture media Dulbecco's modified eagle medium (DMEM)-LG, DMEM-HG, minimum essential medium alpha (MEM α), RPMI-1640, and DMEM/F12 were compared. Cultured BM-eMSCs were collected at day 7 and day 14 to measure cell number and gene expression. Relative gene expression was performed using real-time RT-PCR. Our results showed that MEM α was significantly superior to other media (P < .05) in 14-day culture, enhancing the expression of eMSC genes (ITGB1, CD44 and POU5F1) but depressing differentiation genes (DCN, PPARG and ADIPOQ) in addition to promoting rapid cell proliferation when compared to the other media. We concluded that MEM α was the best media to propagate BM-eMSCs up to 14 days as it supported cell proliferation while maintaining eMSC gene expression. From this, we propose that MEM α may be the most suitable medium for short-term culture of BM-eMSCs for cell transplantation studies.  相似文献   

16.
Reasons for performing study: Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. Objectives: To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. Methods: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT‐qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. Results: High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT‐ and BM‐MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM‐MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. Conclusions: Proliferation ability is different in AT‐MSCs and BM‐MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT‐MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. Potential relevance: To provide data to better understand AT‐MSCs and BM‐MSCs behaviour in vitro.  相似文献   

17.
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.  相似文献   

18.
Del Bue  M.  Ricc&#;  S.  Ramoni  R.  Conti  V.  Gnudi  G.  Grolli  S. 《Veterinary research communications》2008,32(1):51-55
Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.  相似文献   

19.
Reasons for performing study: Autologous cellular therapy products including adipose‐derived stromal vascular fraction (SVF), bone marrow mononuclear cells (BMMNs), cord blood mononuclear cells (CBMNs) and platelet rich plasma are options for treatment of acute orthopaedic lesions while mesenchymal stem cells (MSCs) are culture expanded. These products may contribute to healing by secreting matrix proteins or growth factors, but they may also act on endogenous MSCs to facilitate healing. Objectives: To determine the effects of cell therapy products on MSCs function in vitro. The hypothesis was that cell therapy products promote MSCs functions including proliferation, migration and mediator release. Methods: Fat, bone marrow (BM), cord blood and platelets were obtained from 6 Quarter Horses. The BM‐MSCs and their autologous cell therapy products were co‐incubated in transwells. Mesenchymal stem cells proliferation, migration, gene expression and cytokine concentrations were determined. Results: All cell therapy products increased MSCs proliferation, but SVF induced significantly more proliferation than any other product. Also SVF elicited more MSCs chemotaxis and, along with BMMNs, significantly more MSCs chemoinvasion. Cord blood mononuclear cells stimulated MSCs to produce high concentrations of interleukin‐6 (IL‐6), transforming growth factor‐β1 (TGF‐β1), and prostaglandin E2 (PGE2). Stromal vascular fraction and platelet lysate did not stimulate MSCs but SVF and platelet lysate themselves contained high concentrations of PGE2 and IL‐6 (SVF) and TGF‐β1 (platelet lysate). Conclusions: Autologous cell products variably stimulate MSCs functions with 2 primary patterns apparent. Products either contained preformed mediators that may have intrinsic healing function, or products stimulated MSCs to secrete mediators. Potential relevance: The specific clinical indications for these products may differ to include administration as a sole treatment modality prior to MSCs injection for intrinsic cell and cytokine activity (i.e. SVF) or administration concurrently with MSCs to activate MSCs for treatment of chronic lesions (i.e. CBMNs).  相似文献   

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