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1.
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   

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   

3.
Reasons for performing study: Injury to the superficial digital flexor tendon (SDFT) is common in racing and sport horses and poor tendon regeneration leads to high reinjury rates. Autologous mesenchymal stromal cells (MSCs) are being used clinically to improve tendon regeneration but they have some practical limitations. Embryonic stem cells (ESCs) may overcome these limitations but their fate following injection into the damaged SDFT is unknown. Objective: To inject MSCs and ESCs into distinct areas of damage in the SDFT and monitor their survival over a 3 month period. Methods: MSCs and ESCs expressing different reporter genes were injected into separate sites of mechanically induced damage in SDFTs. Cell survival and distribution were examined post mortem after 10, 30, 60 and 90 days and host immune responses determined. Results: Neither MSCs nor ESCs produced signs of cell‐mediated immune response or tumour formation. ESC survival was high and numbers were maintained at a constant level over 90 days. ESCs were present at all sites of damage. In contrast, MSCs showed <5% survival at 10 days and numbers declined over the course of the experiment. MSCs were detected only at the site into which they were injected. Conclusions: ESCs survived in greater numbers than MSCs in the damaged tendon and did not induce an immune response, or form tumours at the injection sites in the 90 day time period studied. ESCs also demonstrated an ability to migrate to other areas of damage within the same tendon, whereas MSCs did not. Potential relevance: ESCs can be used allogeneically, therefore providing a possible ‘off the shelf’ source of cells for therapeutic use which overcomes the practical limitations of autologous MSCs. Furthermore, MSCs and ESCs have different survival rates and migration patterns in the damaged tendon, suggesting that they may produce different functional effects. This may have clinical relevance to treating tendon injuries in the horse.  相似文献   

4.
Multipotent mesenchymal stromal cells (MSCs) are a promising therapeutic tool for the treatment of equine tendon and other musculoskeletal injuries. While bone marrow is considered the ‘gold standard’ source of these cells, various other tissues contain MSCs with potentially useful features. The aim of this study was to compare clinically relevant characteristics of MSCs derived from bone marrow, umbilical cord blood and tissue and from adipose tissue and tendon. Cell yield, proliferation, migration, tendon marker expression and differentiation into adipocytes, chondrocytes and osteoblasts was assessed, quantified and compared.MSC numbers obtained from adipose, tendon or umbilical cord tissues were 222-fold higher than those obtained from bone marrow or cord blood. Cells derived from tendon and adipose tissues exhibited most rapid proliferation. Osteogenic differentiation was most prominent in MSCs derived from bone marrow, and was weak in MSCs derived from umbilical cord blood and tissue. In contrast, the highest levels of chondrogenic differentiation were observed in MSCs derived from these sources. Collagen 1A2 expression was highest in adipose- and tendon-derived MSCs, while scleraxis expression was highest in cord blood- and in tendon-derived MSCs. The findings indicate that MSCs from different sources display significantly diverse properties that may impact on their therapeutic application.  相似文献   

5.
The goal in the therapeutic use of mesenchymal stem cells (MSCs) in musculoskeletal disease is to harness the regenerative nature of these cells focussing on their potential to grow new tissues and organs to replace damaged or diseased tissue. Laboratory isolation of MSCs is now well established and has recently been demonstrated for equine MSCs. Stem cell science has attracted considerable interest in both the scientific and clinical communities because of its potential to regenerate tissues. Research into the use of MSCs in tissue regeneration in general reflects human medical needs, however, the nature, prevalence and prognosis of superficial digital flexor tendonitis has put equine veterinary science at the forefront of tendon regeneration research. Much has been investigated and learnt but it must be appreciated that in spite of this, the field is still relatively young and both communities must prepare themselves for considerable time and effort to develop the technology into a highly efficient treatments. The promise of functional tissue engineering to replace old parts with new fully justifies the interest. At present, however, it is important to balance the understanding of our current limitations with a desire to progress the technology.  相似文献   

6.
Adult mammalian tissue contains a population of cells known as mesenchymal stem cells (MSC), that possess the capability to secrete regenerative cytokines and to differentiate into specialised cell types. When transplanted to a site of injury MSC embed in damaged tissue and repair and regenerate the tissue by secreting cytokines. The immuno-privileged and immuno-regulatory capabilities of MSC enhance their therapeutic potential not only in autologous but also allogeneic recipients. Studies have demonstrated the beneficial effects of MSC in the treatment of a variety of clinical conditions including osteoarthritis, tendon injuries, and atopic dermatitis in domestic animals. Studies using animal models have shown promising results following MSC or MSC secretion therapy for induced injury in musculoskeletal and nervous systems and some organ diseases. This review describes the stem cell types relevant to regenerative medicine and the procedures used for isolation, identification, expansion, enrichment and differentiation of these cells. We also review the use of MSC in animal models of disease as well as diseases in the clinical veterinary setting.  相似文献   

7.
Locomotor system disorders in equine species, such as tendon lesions, osteoarthritis, or ligament injuries, are some of the most frequent causes of dramatic reduction in horse performance. Traditional therapies are aimed at the inflammatory process and pain, but they do not regenerate normal tendon or ligament matrix and do not reduce re-injured rates. Mesenchymal stem cells have started to use as therapeutic option to repair these injured tissues. Most studies have focused on their isolation, in vitro culture and phenotyping. However, mesenchymal stem cell ultrastructure has been disregarded in the last years. We investigate the ultrastructural characteristics of these cells once differentiated into chondrocytes. Ultrastructural analysis was conducted on suspension cultures of differentiated chondrocytes from bone marrow mesenchymal stem cells by means of transmission electron microscopy. The morphologic characteristics of these cells, their ability to produce the extracellular matrix, and the presence of a single cilium could be indicative of the mesenchymal cells differentiation into chondrocyte phenotype. This study provides essential data to evaluate the degree of suitable phenotypic stability, for these cells can be used with repair purposes.  相似文献   

8.
In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD‐MSCs) to characterize and differentiate them into endothelial‐like cells. AD‐MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony‐forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM‐2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial‐like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD‐MSCs showed their ability to form clones, to differentiate in vitro and the OCT‐4, SOX‐2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial‐like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels‐like structures. This study lays the foundations for future evaluation of the potential AD‐MSCs pro‐angiogenic and therapeutic role.  相似文献   

9.
In the dog, mesenchymal stem cells (MSCs) have been shown to reside in the bone marrow (bone marrow-derived mesenchymal stem cells: BM-MSCs) as well as in the adipose tissue (adipose tissue-derived stem cells: ADSCs). Potential application fields for these multipotent MSCs in small animal practice are joint diseases as MSCs of both sources have shown to possess chondrogenic differentiation ability. However, it is not clear whether the chondrogenic differentiation potential of cells of these two distinct tissues is truly equal. Therefore, we compared MSCs of both origins in this study in terms of their chondrogenic differentiation ability and suitability for clinical application. BM-MSCs harvested from the femoral neck and ADSCs from intra-abdominal fat tissue were examined for their morphology, population doubling time (PDT) and CD90 surface antigen expression. RT-PCR served to assess expression of pluripotency marker Oct4 and early differentiation marker genes. Chondrogenic differentiation ability was compared and validated using histochemistry, transmission electron microscopy (TEM) and quantitative RT-PCR. Both cell populations presented a highly similar morphology and marker expression in an undifferentiated stage except that freshly isolated ADSCs demonstrated a significantly faster PDT than BM-MSCs. In contrast, BM-MSCs revealed a morphological superior cartilage formation by the production of a more abundant and structured hyaline matrix and higher expression of lineage specific genes under the applied standard differentiation protocol. However, further investigations are necessary in order to find out if chondrogenic differentiation can be improved in canine ADSCs using different protocols and/or supplements.  相似文献   

10.
The aim of the present study was to compare canine adipose tissue mesenchymal stem cells cultured under normoxic (20% O2) and not severe hypoxic (7% O2) conditions in terms of marker expression, proliferation rate, differentiation potential and cell morphology. Intra-abdominal fat tissue samples were recovered from 4 dogs and cells isolated from each sample were cultured under hypoxic and normoxic conditions. Proliferation rate and adhesion ability were determined, differentiation towards chondrogenic, osteogenic and adipogenic lineages was induced; the expression of CD44, CD34, DLA-DQA1, DLA-DRA1 was determined by PCR, while flow cytometry analysis for CD90, CD105, CD45 and CD14 was carried out. The morphological study was performed by transmission electron microscopy. Canine AT-MSCs, cultured under different oxygen tensions, maintained their basic biological features. However, under hypoxia, cells were not able to form spheroid aggregates revealing a reduction of their adhesivness. In both conditions, MSCs mainly displayed the same ultrastructural morphology and retained the ability to produce membrane vesicles. Noteworthy, MSCs cultivated under hypoxya revealed a huge shedding of large complex vesicles, containing smaller round-shaped vesicles. In our study, hypoxia partially influences the basic biological properties and the ultrastructural features of canine mesenchymal stem /stromal cells. Further studies are needed to clarify how hypoxia affects EVs production in term of amount and content in order to understand its contribution in tissue regenerative mechanisms and the possible employment in clinical applications. The findings of the present work could be noteworthy for canine as well as for other mammalian species.  相似文献   

11.
The use of adult stem cells in tissue regeneration appears to be a powerful research tool, due to the intrinsic characteristics of these cells, i.e., self-renewal and unlimited capacity for proliferation. In particular, mesenchymal stem cells (MSCs) obtained from bone marrow or peripheral blood can be easily isolated, cultivated, propagated and can be differentiated into several specialized cell types thanks to their plasticity. Among these cells, MSCs can evolve into cardiac cell lineages. Since heart damage leads to the irreversible loss of cardiac function, cell transplantation could be a potential therapy for heart injury. Our laboratory has focused on the purification and expansion of rat and sheep MSCs, their differentiation into cardiomyocytes and their characterisation. Numerous results indicate that MSCs could be promising for therapy, however we need to better understand the biology of stem cells to improve methods for delivery and/or pharmacological activation. These techniques can indeed track engrafted cells and systems to guarantee their safe use.  相似文献   

12.
Background: Adult mesenchymal stem cells(MSCs) can be conveniently sampled from bone marrow, peripheral blood, muscle, adipose and connective tissue, harvested from various species, including, rodents, dogs, cats, horses,sheep, goats and human beings. The MSCs isolated from adult tissues vary in their morphological and functional properties. These variations are further complicated when cells are expanded by passaging in culture. These differences and changes in MSCs must be considered prior to their application in the clinic or in a basic research study. Goats are commonly used as animal models for bone tissue engineering to test the potential of stem cells for bone regeneration. As a result, goat MSCs isolated from bone marrow or adipose tissue should be evaluated using in vitro assays, prior to their application in a tissue engineering project.Results: In this study, we compared the stem cell properties of MSCs isolated from goat bone marrow and adipose tissue. We used quantitative and qualitative assays with a focus on osteogenesis, including, colony forming unit, rate of cell proliferation, tri-lineage differentiation and expression profiling of key signal transduction proteins to compare MSCs from low and high passages. Primary cultures generated from each source displayed the stem cell characteristics,with variations in their osteogenic potentials. Most importantly, low passaged bone marrow MSCs displayed a significantly higher and superior osteogenic potential, and hence, will be the preferred choice for bone tissue engineering in future in vivo experiments. In the bone marrow MSCs, this process is potentially mediated by the p38 MAPK pathway. On the other hand, osteogenic differentiation in the adipose tissue MSCs may involve the p44/42 MAPK pathway.Conclusions: Based on these data, we can conclude that bone marrow and fat-derived MSCs undergo osteogenesis via two distinct signaling pathways. Even though the bone marrow MSCs are the preferred source for bone tissue engineering, the adipose tissue MSCs are an attractive alternative source and undergo osteo-differentiation differently from the bone marrow MSCs and hence, might require a cell-based enhancer/inducer to improve their osteogenic regenerative capacity.  相似文献   

13.
Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells, which can differentiation into cells of connective tissue and neural lineages. This study investigated the potential for neuronal differentiation of red fluorescent protein (RFP)-transgenic cat UCB-derived MSCs. The cells were cultured in pre-induction medium for 24 hr and in neuronal-induction medium for 72 hr. Immunofluorescent staining showed that 6.85% of the total cells were beta III-tubulin-positive, 3.37% were neurofilament light (NF-L)-positive and 7.04% were neurofilament medium (NF-M)-positive. A beta III-tubulin band was detected by western blot analysis. Our results demonstrate that RFP-transgenic UCB-derived MSCs can be differentiated into neuronal cells in vitro. Thus, RFP-transgenic MSCs could provide alternative tracing material for stem cell transplantation.  相似文献   

14.
The objective of this study was to graft autologous mesenchymal stem cells (MSCs) at the site of surgical repair of a soft palate defect in an adult horse in an attempt to improve wound healing and to investigate whether the transplanted MSCs would integrate into the soft palate structure and participate in regeneration. Bone marrow was collected from an adult horse with a full-thickness soft palate defect. The MSCs were isolated, cultured in monolayers, and labeled with 5-bromo-2-desoxymidine (BrdU) and chloromethylbenzamido-DiI-derived (cm-DiI) before transplantation. The soft palate defect was repaired by mandibular symphysiotomy, and the labeled MSCs were injected into the repaired soft palate. Postmortem examination revealed that 90% of the soft palate defect had been sutured. Staining by BrdU and cm-DiI was intense in the soft palate tissue. Labeled MSCs were detected in tissue slices from the injection sites. The cells were organized in a manner similar to that in native soft palate tissue, indicating successful engraftment.  相似文献   

15.
Mesenchymal stem cells have demonstrated their potentiality for therapeutic use in treating diseases or repairing damaged tissues. However, in some cases, the results of clinical trials have been disappointing or have not worked out as well as hoped. These disappointing results can be attributed to an inadequate or insufficient preclinical study. For medical and surgical purposes, the similarities between the anatomy of pig and human make this animal an attractive preclinical model. In this sense, for mesenchymal stem cell-based therapy, it is strongly necessary to have well characterized animal-derived mesenchymal stem cell lines to validate preclinical effectiveness of these cells. In this work, porcine mesenchymal stem cells (pMSCs) were isolated from bone marrow, adipose tissue and peripheral blood and compared in terms of differentiation potential, cell surface markers and gene expression. Our results demonstrated that the isolation and in vitro expansion protocols were feasible and effective. The data presented in this work are relevant because they provide an extensive phenotypic characterization; genetic study and differentiation behavior of the most commonly used stem cell lines for clinical practices. These pMSCs are widely available to scientists and could be a valuable tool to evaluate the safety and efficacy of adoptively transferred cells.  相似文献   

16.
Currently, mesenchymal stem cells (MSCs) are used in veterinary clinical applications. Bone marrow and adipose tissue are the most common sources of stem cells derived from adult animals. However, cord blood which is collected non‐invasively is an alternative source of stem cells other than bone marrow and adipose tissue. Moreover, high availability and lower immunogenicity of umbilical cord blood (UCB) haematopoietic stem cells compared to other sources of stem cell therapy such as bone marrow have made them a considerable source for cell therapy, but MSCs is not highly available in cord blood and their immunogenicity is poorly understood. In this study, the cells with spindle morphology from 7 of 9 bovine UCB samples were isolated and cultured. These mesenchymal stromal cells were successfully differentiated to osteocytes, chondrocytes and adipocytes. In addition, Oct‐4 and SH3 were determined by RT‐PCR assay. It is the first report of isolation, culture, characterization and differentiation of bovine umbilical stem cells.  相似文献   

17.
Aim of the present study was in vitro expansion and characterization of caprine wharton’s jelly derived mesenchymal stem cells (cWJ-MSCs) to investigate their tissue healing potential in xenogenic animal model. Plastic adherent fibroblastoid cell populations with distinctive homogeneous morphology were isolated from caprine wharton’s jelly explants. These wharton’s jelly derived cells were found positive for the surface markers CD-73, STRO-1 and CD-105, whereas they were negative for hematopoetic stem cell marker CD-34. In vitro cultured cWJ-MSCs also showed differentiation properties into osteogenic, adipogenic and chondrogenic lineages as demonstrated by von Kossa, Oil Red- O and Alcian blue staining respectively, which was further confirmed and quantified by flow cytometric analysis. Furthermore, these well characterized cWJ-MSCs were evaluated for the wound-healing potential in full-thickness skin wounds in rabbit model for 28 days. Caprine WJ- MSCs treated skin wounds showed significantly (P?<?0.05) higher percentage of wound contraction especially at the 21st day post transplantation when compared to PBS treated control group animals. Further, we observed better healing potential of cWJ-MSCs in terms of histo-morphological evaluation, epithelialisation and collagenization with matured vascularization stage by day 28 as compared to control. In conclusion, cWJ- MSCs provide an alternative inexhaustible source of mesenchymal stem cells and also unravel new perspectives pertaining to the therapeutic use of these cells in different species.  相似文献   

18.
Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III β tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.  相似文献   

19.
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   

20.
Repair of injured soft and hard tissues in horses can benefit greatly from the use of regenerative therapies with mesenchymal stem cells (MSC). Vitamin-C and platelet-rich-plasma had been used for in vitro differentiation of MSC. This study was aimed to evaluate the effect of vitamin-C, platelet-rich-plasma and their combination on the in vitro differentiation of adipose horse MSC. We isolated MSC from horse fat and differentiated them in vitro into osteogenic and chondrogenic lineages, as demonstrated by specific staining and RT-qPCR of selected genes. Combining vitamin-C and plasma-rich-platelet positively affected the ability of MSC to differentiate in vitro into mesodermal lineages during 14 days of culture; this effect was not as marked when differentiation was attempted for 21 days. This provides valuable information on the effect of combined use of these molecules in regenerative therapies and their potential application along stem cells for lesions of musculoskeletal tissue in sport horses.  相似文献   

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