Equine Corneal Wound Healing Using Mesenchymal Stem Cell Secretome: Case Report.

Corneal ulcers are a common and potentially vision-threatening condition in horses that can be challenging to treat with conventional therapies alone. This case report describes the successful treatment of a non-healing corneal ulcer in a 28-year-old Hispano-Bretón mare using the secretome derived from adipose tissue-derived mesenchymal stem cells (ASCs). Despite initial treatment with antibiotics, anti-inflammatory drugs, and surgical debridement, the corneal ulcer failed to heal properly, exhibiting persistent epithelial defects and stromal complications. As an alternative regenerative approach, the ASC secretome, a rich source of trophic factors, cytokines, and extracellular vesicles, was topically administered to the affected eye. Remarkably, within one week of secretome treatment, the clinical signs of blepharospasm and epiphora resolved, and the corneal ulcer exhibited complete re-epithelialization, regained transparency, and reduced neovascularization. No recurrence was observed during the 1.5-year follow-up period. This case highlights the potential therapeutic benefits of the ASC secretome in promoting corneal wound healing and suggests its promise as a novel cell-free therapy for treating refractory corneal ulcers in horses.

Application of adipose-derived mesenchymal stem cells in an in vivo model of peripheral nerve damage.

Background: Neuropathic pain is one of the most difficult to treat chronic pain syndromes. It has significant effects on patients' quality of life and substantially adds to the burden of direct and indirect medical costs. There is a critical need to improve therapies for peripheral nerve regeneration. The aim of this study is to address this issue by performing a detailed analysis of the therapeutic benefits of two treatment options: adipose tissue derived-mesenchymal stem cells (ASCs) and ASC-conditioned medium (CM). Methods: To this end, we used an in vivo rat sciatic nerve damage model to investigate the molecular mechanisms involved in the myelinating capacity of ASCs and CM. Furthermore, effect of TNF and CM on Schwann cells (SCs) was evaluated. For our in vivo model, biomaterial surgical implants containing TNF were used to induce peripheral neuropathy in rats. Damaged nerves were also treated with either ASCs or CM and molecular methods were used to collect evidence of nerve regeneration. Post-operatively, rats were subjected to walking track analysis and their sciatic functional index was evaluated. Morphological data was gathered through transmission electron microscopy (TEM) of sciatic nerves harvested from the experimental rats. We also evaluated the effect of TNF on Schwann cells (SCs) in vitro. Genes and their correspondent proteins associated with nerve regeneration were analyzed by qPCR, western blot, and confocal microscopy. Results: Our data suggests that both ASCs and CM are potentially beneficial treatments for promoting myelination and axonal regeneration. After TNF-induced nerve damage we observed an upregulation of c-Jun along with a downregulation of Krox-20 myelin-associated transcription factor. However, when CM was added to TNF-treated nerves the opposite effect occurred and also resulted in increased expression of myelin-related genes and their corresponding proteins. Conclusion: Findings from our in vivo model showed that both ASCs and CM aided the regeneration of axonal myelin sheaths and the remodeling of peripheral nerve morphology.

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.

Isolation and characterization of exosomes from adipose tissue-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are the subject of intense research as they are a potential therapeutic tool for several clinical applications. The new MSCs action models are focused on the use of MSC-derived secretome which contains several growth factors, cytokines, microRNAs, and extracellular vesicles such as exosomes. Exosomes have recently emerged as a component with great potential involved as mediators in cellular communication. The isolation and identification of exosomes has made it possible for them to be used in cell-free therapies. The purposes of this study are: (i) to detect exosomes released into adipose-derived MSC conditioned cell culture medium, (ii) to identify exosome morphology, and (iii) to carry out a complete characterization of said exosomes. Moreover, it is aimed at determining which method for exosome isolation would be best to use. Precipitation has been identified as a highly useful method of exosome isolation since it provides higher efficiency and purity values than other methods. A broad characterization of the exosomes present in the MSC-conditioned medium was also carried out. This work fills a gap in the existing literature on bioactive molecules which have attracted a great deal of interest due to their potential use in cellular therapies.

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.