Isolation and morphological characterization of equine mesenchymal stem cells from harvested adipose tissue and bone marrow and stably transfected with green fluorescent protein.

OBJECTIVE: To characterize the ultrastructure of mesenchymal stem cells (MSCs) that were harvested from the adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) of horses and transfected with green fluorescent protein. SAMPLE: MSCs from adipose tissue and bone marrow of 6 adult female Hispano-Bretón horses. PROCEDURES: Harvested equine MSCs were cultivated and transfected with green fluorescent protein, and the immunophenotypes of the MSCs were characterized by use of anti-CD90 and anti-CD105 monoclonal antibodies. When stable transfection of MSCs was achieved, the morphological and ultrastructural characteristics of transfected and nontransfected AT-MSCs and BM-MSCs were compared with electron microscopy. RESULTS: The protocols for transfection and subsequent isolation of transfected cells with use of G418 were suitable for obtaining transfected MSCs. Transfection efficiency was 5% in AT-MSCs and 4% in BM-MSCs. Characterization of transfected and nontransfected MSCs revealed that they share immunocytochemical and morphological profiles. Expression of CD90 was significantly higher for transfected versus nontransfected AT-MSCs (97% vs 92%). Expression of CD105 was significantly lower for transfected versus nontransfected BM-MSCs (85% vs 94%). Transfected BM-MSCs had differences in organelles, compared with the other cell types, specifically including most commonly the rough endoplasmic reticulum with dilated cisternae and mitochondria. CONCLUSION AND CLINICAL RELEVANCE: These findings contribute to the knowledge base of the characteristics of equine AT-MSCs and BM-MSCs and of transfected versus nontransfected equine MSCs. The data provided a valuable starting point for researchers wishing to further study the morphological characteristics of equine MSCs.

Effect of mesenchymal stem cells combined with chondroitin sulfate in an in vitro model of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease affecting the whole joint structure. The specific molecules responsible for the inflammatory processes involved in the development of OA have been the focus of many studies. Adipose tissue-derived mesenchymal stem cells (ASCs) constitute a promising cell-based therapy which could be used as an alternative to or in combination with drug therapies. Chondroitin sulfate (CS) plays a protective role in the joint by decreasing concentrations of pro-inflammatory cytokines and therefore has an important part in moderating chondrocyte metabolism. The aim of this study is to use an in vitro model of OA to evaluate the combined effectiveness of CS and ASCs as a treatment. We give a detailed discussion of the roles of cytokines and other key molecules involved in OA. In addition, we report the effects of treating inflamed chondrocytes with ASCs and CS on the expression of specific cartilage genes. Findings show that both treatments reduced expression of all genes associated with the pro-inflammatory cytokines we analyzed. However, we saw no increase in the expression of the specific genes encoding for cartilage matrix proteins, such as collagen type II and aggrecan. This study shows the effectiveness of combining ASCs and CS in the treatment of OA.

Immunomodulation of mesenchymal stem cells in discogenic pain.

BACKGROUND CONTEXT: Back pain is a highly prevalent health problem in the world today and has a great economic impact on health-care budgets. Intervertebral disc (IVD) degeneration has been identified as a main cause of back pain. Inflammatory cytokines produced by macrophages or disc cells in an inflammatory environment play an important role in painful progressive degeneration of IVD. Mesenchymal stem cells (MSCs) have shown to have immunosuppressive and anti-inflammatory properties. Mesenchymal stem cells express a variety of chemokines and cytokines receptors having tropism to inflammation sites. PURPOSE: This study aimed to develop an in vitro controlled and standardized model of inflammation and degeneration of IVD with rat cells and to evaluate the protective and immunomodulatory effect of conditioned medium (CM) from the culture of MSCs to improve the conditions presented in herniated disc and discogenic pain processes. STUDY DESIGN: This is an experimental study. METHODS: In this study, an in vitro model of inflammation and degeneration of IVD has been developed, as well as the effectiveness of CM from the culture of MSCs. RESULTS: Conditioned medium from MSCs downregulated the expression of various proinflammatory cytokines produced in the pathogenesis of discogenic pain such as interleukin (IL)-1ß, IL-6, IL-17, and tumor necrosis factor (TNF). CONCLUSION: Mesenchymal stem cells represent a promising alternative strategy in the treatment of IVD degeneration inasmuch as there is currently no treatment which leads to a complete remission of long-term pain in the absence of drugs.

Assessment of regeneration in meniscal lesions by use of mesenchymal stem cells derived from equine bone marrow and adipose tissue.

OBJECTIVE To assess the ability to regenerate an equine meniscus by use of a collagen repair patch (scaffold) seeded with mesenchymal stem cells (MSCs) derived from bone marrow (BM) or adipose tissue (AT). SAMPLE 6 female Hispano-Breton horses between 4 and 7 years of age; MSCs from BM and AT were obtained for the in vitro experiment, and the horses were subsequently used for the in vivo experiment. PROCEDURES Similarities and differences between MSCs derived from BM or AT were investigated in vitro by use of cell culture. In vivo assessment involved use of a meniscus defect and implantation on a scaffold. Horses were allocated into 2 groups. In one group, defects in the medial meniscus were treated with MSCs derived from BM, whereas in the other group, defects were treated with MSCs derived from AT. Defects were created in the contralateral stifle joint but were not treated (control samples). RESULTS Both types of MSCs had universal stem cell characteristics. For in vivo testing, at 12 months after treatment, treated defects were regenerated with fibrocartilaginous tissue, whereas untreated defects were partially repaired or not repaired. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MSCs derived from AT could be a good alternative to MSCs derived from BM for use in regenerative treatments. Results also were promising for a stem cell-based implant for use in regeneration in meniscal lesions. IMPACT FOR HUMAN MEDICINE Because of similarities in joint disease between horses and humans, these results could have applications in humans.