Chondrogenesis of canine and equine mesenchymal stem cells.

Naomie Tidd

    Research output: ThesisMasters Thesis

    Abstract

    Repair of the unique and highly specialised structure of articular cartilage presents a significant biological challenge for health professionals and veterinarians. Current methodologies frequently result in a short-term fix, with inferior mechanical properties to native articular cartilage, an unsatisfactory outcome particularly to younger patients. The work described in this thesis examined in vitro, the suitability of mesenchymal stem cells (MSCs) for cartilage regenerative therapies from two animals in which cartilage damage can have a debilitating effect, the horse and dog, with the aim of applying these to further in vivo applications. Adipose-derived canine and equine MSCs were assessed for their in vitro capacity to differentiate into chondrocytes. A clear species-specific difference was observed, with cell senescence recognised as a likely significant factor in preventing chondrogenic differentiation of canine MSCs. This suggests that optimisation of the current protocols for MSC expansion and differentiation would be essential in order to utilise these cell types for regenerative therapies.In an effort to improve the chondrogenic and therapeutic capacity of MSCs, an in vitro gene expression vector was identified and validated, for ex vivo and in vivo delivery of Sox9 to MSCs, a gene essential to initiation and regulation of cartilage formation. Although lentiviral vectors offered sustained expression of transgene over several passages in both species, lentiviral-mediated Sox9 delivery to equine MSCs failed to improve chondrogenic outcomes in vitro. The episomal minicircle vector has key biosafety advantages over viral vector systems for regenerative therapies, and offered high transfection efficiency in canine MSCs. Transfection of the minicircle-Sox9 vector into canine MSCs was also successful with Sox9 immunostaining confirming protein expression in the target cell lines. The results of this study demonstrate the application and efficacy of a novel non-viral expression vector in MSCs from non-traditional laboratory species with a veterinary application. These results indicate the potential usefulness for gene-directed veterinary regenerative therapies in both canine and equines for treatment of cartilage injury and repair.
    Original languageEnglish
    QualificationMaster of Applied Science
    Awarding Institution
    • Charles Sturt University
    Place of PublicationAustralia
    Publisher
    Publication statusPublished - 2015

    Fingerprint

    Chondrogenesis
    Mesenchymal Stromal Cells
    Horses
    Canidae
    Cartilage
    Articular Cartilage
    Transfection
    Therapeutics
    Veterinarians
    Cell Aging
    Essential Genes
    Chondrocytes
    Cell- and Tissue-Based Therapy
    Transgenes
    Cell Differentiation
    Dogs
    Gene Expression

    Cite this

    Tidd, N. (2015). Chondrogenesis of canine and equine mesenchymal stem cells. Australia: Charles Sturt University.
    Tidd, Naomie. / Chondrogenesis of canine and equine mesenchymal stem cells.. Australia : Charles Sturt University, 2015.
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    title = "Chondrogenesis of canine and equine mesenchymal stem cells.",
    abstract = "Repair of the unique and highly specialised structure of articular cartilage presents a significant biological challenge for health professionals and veterinarians. Current methodologies frequently result in a short-term fix, with inferior mechanical properties to native articular cartilage, an unsatisfactory outcome particularly to younger patients. The work described in this thesis examined in vitro, the suitability of mesenchymal stem cells (MSCs) for cartilage regenerative therapies from two animals in which cartilage damage can have a debilitating effect, the horse and dog, with the aim of applying these to further in vivo applications. Adipose-derived canine and equine MSCs were assessed for their in vitro capacity to differentiate into chondrocytes. A clear species-specific difference was observed, with cell senescence recognised as a likely significant factor in preventing chondrogenic differentiation of canine MSCs. This suggests that optimisation of the current protocols for MSC expansion and differentiation would be essential in order to utilise these cell types for regenerative therapies.In an effort to improve the chondrogenic and therapeutic capacity of MSCs, an in vitro gene expression vector was identified and validated, for ex vivo and in vivo delivery of Sox9 to MSCs, a gene essential to initiation and regulation of cartilage formation. Although lentiviral vectors offered sustained expression of transgene over several passages in both species, lentiviral-mediated Sox9 delivery to equine MSCs failed to improve chondrogenic outcomes in vitro. The episomal minicircle vector has key biosafety advantages over viral vector systems for regenerative therapies, and offered high transfection efficiency in canine MSCs. Transfection of the minicircle-Sox9 vector into canine MSCs was also successful with Sox9 immunostaining confirming protein expression in the target cell lines. The results of this study demonstrate the application and efficacy of a novel non-viral expression vector in MSCs from non-traditional laboratory species with a veterinary application. These results indicate the potential usefulness for gene-directed veterinary regenerative therapies in both canine and equines for treatment of cartilage injury and repair.",
    author = "Naomie Tidd",
    year = "2015",
    language = "English",
    publisher = "Charles Sturt University",
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    school = "Charles Sturt University",

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    Tidd, N 2015, 'Chondrogenesis of canine and equine mesenchymal stem cells.', Master of Applied Science, Charles Sturt University, Australia.

    Chondrogenesis of canine and equine mesenchymal stem cells. / Tidd, Naomie.

    Australia : Charles Sturt University, 2015.

    Research output: ThesisMasters Thesis

    TY - THES

    T1 - Chondrogenesis of canine and equine mesenchymal stem cells.

    AU - Tidd, Naomie

    PY - 2015

    Y1 - 2015

    N2 - Repair of the unique and highly specialised structure of articular cartilage presents a significant biological challenge for health professionals and veterinarians. Current methodologies frequently result in a short-term fix, with inferior mechanical properties to native articular cartilage, an unsatisfactory outcome particularly to younger patients. The work described in this thesis examined in vitro, the suitability of mesenchymal stem cells (MSCs) for cartilage regenerative therapies from two animals in which cartilage damage can have a debilitating effect, the horse and dog, with the aim of applying these to further in vivo applications. Adipose-derived canine and equine MSCs were assessed for their in vitro capacity to differentiate into chondrocytes. A clear species-specific difference was observed, with cell senescence recognised as a likely significant factor in preventing chondrogenic differentiation of canine MSCs. This suggests that optimisation of the current protocols for MSC expansion and differentiation would be essential in order to utilise these cell types for regenerative therapies.In an effort to improve the chondrogenic and therapeutic capacity of MSCs, an in vitro gene expression vector was identified and validated, for ex vivo and in vivo delivery of Sox9 to MSCs, a gene essential to initiation and regulation of cartilage formation. Although lentiviral vectors offered sustained expression of transgene over several passages in both species, lentiviral-mediated Sox9 delivery to equine MSCs failed to improve chondrogenic outcomes in vitro. The episomal minicircle vector has key biosafety advantages over viral vector systems for regenerative therapies, and offered high transfection efficiency in canine MSCs. Transfection of the minicircle-Sox9 vector into canine MSCs was also successful with Sox9 immunostaining confirming protein expression in the target cell lines. The results of this study demonstrate the application and efficacy of a novel non-viral expression vector in MSCs from non-traditional laboratory species with a veterinary application. These results indicate the potential usefulness for gene-directed veterinary regenerative therapies in both canine and equines for treatment of cartilage injury and repair.

    AB - Repair of the unique and highly specialised structure of articular cartilage presents a significant biological challenge for health professionals and veterinarians. Current methodologies frequently result in a short-term fix, with inferior mechanical properties to native articular cartilage, an unsatisfactory outcome particularly to younger patients. The work described in this thesis examined in vitro, the suitability of mesenchymal stem cells (MSCs) for cartilage regenerative therapies from two animals in which cartilage damage can have a debilitating effect, the horse and dog, with the aim of applying these to further in vivo applications. Adipose-derived canine and equine MSCs were assessed for their in vitro capacity to differentiate into chondrocytes. A clear species-specific difference was observed, with cell senescence recognised as a likely significant factor in preventing chondrogenic differentiation of canine MSCs. This suggests that optimisation of the current protocols for MSC expansion and differentiation would be essential in order to utilise these cell types for regenerative therapies.In an effort to improve the chondrogenic and therapeutic capacity of MSCs, an in vitro gene expression vector was identified and validated, for ex vivo and in vivo delivery of Sox9 to MSCs, a gene essential to initiation and regulation of cartilage formation. Although lentiviral vectors offered sustained expression of transgene over several passages in both species, lentiviral-mediated Sox9 delivery to equine MSCs failed to improve chondrogenic outcomes in vitro. The episomal minicircle vector has key biosafety advantages over viral vector systems for regenerative therapies, and offered high transfection efficiency in canine MSCs. Transfection of the minicircle-Sox9 vector into canine MSCs was also successful with Sox9 immunostaining confirming protein expression in the target cell lines. The results of this study demonstrate the application and efficacy of a novel non-viral expression vector in MSCs from non-traditional laboratory species with a veterinary application. These results indicate the potential usefulness for gene-directed veterinary regenerative therapies in both canine and equines for treatment of cartilage injury and repair.

    M3 - Masters Thesis

    PB - Charles Sturt University

    CY - Australia

    ER -

    Tidd N. Chondrogenesis of canine and equine mesenchymal stem cells.. Australia: Charles Sturt University, 2015.