Isolation and characterization of bone marrow-derived equine mesenchymal stem cells

ArticleinAmerican Journal of Veterinary Research 68(10):1095-105 · November 2007with31 Reads
DOI: 10.2460/ajvr.68.10.1095 · Source: PubMed
To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. 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. 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. 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.
    • "Equine mesenchymal stem cells (EMSCs) were isolated from bone marrow1234, adipose tissue [5], peripheral blood [1] and cord blood [6,7]. Currently, there is an increasing interest in the investigation of adult extra-embryonic tissues such as fetal adnexa (amnion, amniotic fl uid and Wharton jelly)8910 due to easy collection aft er birth of the foal. "
    Full-text · Article · Nov 2015
    • "Musculoskeletal injuries are amongst the leading causes of early retirement or euthanasia of race, pleasure, and working horses (Arnhold et al. 2007). Bone fractures commonly occur and prove to be very serious injuries, due to the poor regenerative capacity of damaged cartilage, tendons, and ligaments. "
    [Show abstract] [Hide abstract] ABSTRACT: We investigated the applicability of single layer paper-based scaffolds for the three-dimensional (3D) growth and osteogenic differentiation of equine adipose-derived stem cells (EADSC), with comparison against conventional two-dimensional (2D) culture on polystyrene tissue culture vessels. Viable culture of EADSC was achieved using paper-based scaffolds, with EADSC grown and differentiated in 3D culture retaining high cell viability (>94 %), similarly to EADSC in 2D culture. Osteogenic differentiation of EADSC was significantly enhanced in 3D culture, with Alizarin Red S staining and quantification demonstrating increased mineralisation (p < 0.0001), and an associated increase in expression of the osteogenic-specific markers alkaline phosphatase (p < 0.0001), osteopontin (p < 0.0001), and runx2 (p < 0.01). Furthermore, scanning electron microscopy revealed a spherical morphology of EADSC in 3D culture, compared to a flat morphology of EADSC in 2D culture. Single layer paper-based scaffolds provide an enhanced environment for the in vitro 3D growth and osteogenic differentiation of EADSC, with high cell viability, and a spherical morphology.
    Full-text · Article · Jul 2015
    • "However, unlike for human MSC, variable identification of CD73 and CD105 on MSC has precluded establishment of a consensus panel for horse MSC [9][10][11][12][13][14][15][16][17] . In addition, several investigators reported expression of MHC-II, CD31, CD34, CD45 and CD79a to be low or absent [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. Hence, surface antigens characteristic of equine MSC have neither been clearly established nor confirmed with quantitative gene expression assays, although clinical application of such cells has been widely implemented [6]. "
    [Show abstract] [Hide abstract] ABSTRACT: Multipotent mesenchymal stromal cells (MSC) have attracted interest for their cytotherapeutic potential, partly due to their immunomodulatory abilities. The aim of this study was to test the robustness of our equine cord blood (CB) MSC isolation protocol, to characterize the CB-MSC before and after cryopreservation, and to evaluate their immunosuppressive phenotype. We hypothesized that MSC can be consistently isolated from equine CB, have unique and reproducible marker expression and in vitro suppress lymphoproliferation. Preliminary investigation of constitutive cytoplasmic Toll-like receptor (TLR) 3 and 4 expression was also preformed due to their possible association with anti- or pro-inflammatory MSC phenotypes, respectively. Surface markers were assessed for antigen and mRNA expression by flow cytometry and quantitative polymerase chain reaction (qPCR). Immunomodulatory properties were evaluated in mixed lymphocyte reaction assays, and TLR3 and TLR4 expression were measured by qPCR and immunocytochemistry (ICC). CB-MSC were isolated from each off nine cord blood samples. CB-MSC highly expressed CD29, CD44, CD90, and lacked or had low expression of major histocompatibility complex (MHC) class I, MHC-II, CD4, CD8, CD11a/18 and CD73 before and after cryopreservation. CB-MSC suppressed in vitro lymphoproliferation and constitutively expressed TLR4. Our findings confirmed CB as a reliable MSC source, provides an association of surface marker phenotype and mRNA expression and suggest anti-inflammatory properties of CB-MSC. The relationship between TLRs and lymphocyte function warrants further investigation.
    Full-text · Article · Apr 2015
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