Isolation, characterisation and differentiation of canine adult stem cells

Cardiac and orthopaedic diseases are significant causes of morbidity and mortality in dogs and are therefore critical areas for veterinary research. More information regarding the pathophysiology of these diseases, and the development of novel therapeutics are sorely required and adult stem cells (ASCs) are a promising source of cells for both investigation of these diseases in vitro and also potentially therapeutics in the longer term. ASCs are a readily available source of multipotent cells which bypass the ethical issues surrounding embryonic stem (ES) cells. ASCs have been described in several tissues of the body, and typically differentiate along specific cellular routes related to original source location. This thesis investigates whether ASCs can be isolated and cultured from the dog from two specific locations; cardiac, producing cardiac stem cells (CSCs); and the bone marrow, producing mesenchymal stem cells (MSCs). These cell sources will be extensively characterised at their baseline for morphology, culture behaviour and gene marker expression. Following characterisation each cell source will be subjected to differentiation techniques to examine canine ASC multipotent differentiation potential. CSCs were isolated from cultured atrial cardiac explant tissue taken from dogs post-mortem, with owners’ consent. These cells were able to survive successive passages in serum free media and formed large spherical cell clusters, termed ‘cardiospheres’. CSCs were capable of clonal expansion under controlled culture conditions, demonstrating their ability for self-renewal. Characterisation of these cells demonstrated the expression of CSC markers; c-Kit, GATA 4 and Flk-1 and no expression of cardiac lineage markers including cardiac troponin T and I, Nkx2.5, the cardiac ryanodine receptor and the β1-adrenergic receptor. Primary canine MSCs were isolated from bone marrow aspirates using ficoll separation and cultured on tissue culture plastic. Canine MSCs closely resembled MSCs described from other species, such as the human and mouse, and were found to express CD44 and STRO-1 and were negative for CD34 and CD45. CSCs and MSCs were exposed to published cardiac directed differentiation protocols and differentiation then analysed using cellular morphology and gene expression. Canine CSCs appeared to differentiate partially along cardiac lineages with upregulation of cardiac troponin T and Nkx2.5, and down regulation of c-Kit and endothelial lineage markers. Canine MSCs demonstrated some morphological changes during cardiac differentiation, and demonstrated up-regulation of Nkx2.5 and Flk-1 but no significant alteration in other markers examined. This suggested that cardiac directed differentiation was not as successful with canine MSCs compared to CSCs and conflicting with published data using rodent MSC models. Murine MSCs were used as a positive control cell line for cardiac directed differentiation, based upon published literature. Critically there were key marker expression differences between baseline murine and canine MSCs, including the expression of cardiac markers such as cardiac troponin T and I, and the Ryanodine receptor. Furthermore, expression analysis of cardiac genes changed with time in culture and passage number and no significant alteration was seen when cells were subjected to the cardiac differentiation protocol; thereby bringing into question the data regarding successful cardiac differentiation using murine MSCs. Canine MSCs were further differentiated toward a chondrocyte lineage to investigate the use of MSCs for orthopaedic research. Canine MSCs were successfully differentiated toward articular type cartilage, with demonstration of extracellular matrix secretions, an upregulation of collagen type II with downregulation of collagen type I and the development of SOX9 expression in differentiated cells. This thesis builds the groundwork for future ASC research in the dog. Successful isolation and culture of two ASC sources from the dog is demonstrated. Cardiac and cartilage directed differentiation was successful using primary sourced cells, but differentiation was found to be limited to highly specific routes for each stem cell source. The results presented here highlight the importance of analysing baseline stem cells extensively prior to differentiation and in particular, before making comparisons between cell populations isolated from different locations and species

First report of Lyme borreliosis leading to cardiac bradydysrhythmia in two cats

Case series summary:Two cats were presented for investigation of bradyarrhythmia detected by their referring veterinarians during routine examination. Both cats had extensive investigations, including haematology, serum biochemistry with electrolytes and thyroxine concentrations, systolic blood pressure measurement, echocardiography, electrocardiography and infectious disease testing. Infectious disease testing included serology for Toxoplasma gondii, Ehrlichia canis, Anaplasma phagocytophilum and Borrelia burgdorferi, and PCR for B burgdorferi antigen in both cats. Case 1 was also assessed by PCR for Bartonella henselae antigen and case 2 was assessed for Dirofilaria immitis by serology. All infectious disease tests, other than for B burgdorferi, were negative. Case 1 was diagnosed with Lyme carditis based on marked bradydysrhythmia, positive B burgdorferi serology, a structurally normal heart and clinical resolution with appropriate treatment with a 4-year follow-up. Case 2 was diagnosed with Lyme carditis based on marked bradydysrhythmia and positive B burgdorferi PCR; however, this cat had structural heart disease that did not resolve with treatment. Relevance and novel information:This small case series describes two B burgdorferi positive cats presenting with newly diagnosed cardiac abnormalities consistent with those found in humans and dogs with Lyme carditis. Both cats were asymptomatic as perceived by their owners; the arrhythmia was detected by their veterinarians

Vasovagal tonus index (VVTI) as an indirect assessment of remission status in canine multicentric lymphoma undergoing multi-drug chemotherapy

Vasovagal tonus index (VVTI) is an indirect measure of heart rate variability and may serve as a marker of disease severity. Higher heart rate variability has predicted lower tumour burden and improved survival in humans with various tumour types. The purpose of this pilot study was to evaluate VVTI as a biomarker of remission status in canine lymphoma. The primary hypothesis was that VVTI would be increased in dogs in remission compared to dogs out of remission. Twenty-seven dogs were prospectively enrolled if they had a diagnosis of intermediate to high-grade lymphoma and underwent multidrug chemotherapy. Serial electrocardiogram data were collected under standard conditions and relationships between VVTI, remission status and other clinical variables were evaluated. VVTI from dogs in remission (partial or complete) did not differ from dogs with fulminant lymphoma (naive or at time of relapse). Dogs in partial remission had higher VVTI than dogs in complete remission (p = 0.021). Higher baseline VVTI was associated with higher subsequent scores (p < 0.001). VVTI also correlated with anxiety level (p = 0.03). Based on this pilot study, VVTI did not hold any obvious promise as a useful clinical biomarker of remission status. Further investigation may better elucidate the clinical and prognostic utility of VVTI in dogs with lymphoma

Isolation, characterisation and differentiation of canine adult stem cells

Cardiac and orthopaedic diseases are significant causes of morbidity and mortality in dogs and are therefore critical areas for veterinary research. More information regarding the pathophysiology of these diseases, and the development of novel therapeutics are sorely required and adult stem cells (ASCs) are a promising source of cells for both investigation of these diseases in vitro and also potentially therapeutics in the longer term. ASCs are a readily available source of multipotent cells which bypass the ethical issues surrounding embryonic stem (ES) cells. ASCs have been described in several tissues of the body, and typically differentiate along specific cellular routes related to original source location. This thesis investigates whether ASCs can be isolated and cultured from the dog from two specific locations; cardiac, producing cardiac stem cells (CSCs); and the bone marrow, producing mesenchymal stem cells (MSCs). These cell sources will be extensively characterised at their baseline for morphology, culture behaviour and gene marker expression. Following characterisation each cell source will be subjected to differentiation techniques to examine canine ASC multipotent differentiation potential. CSCs were isolated from cultured atrial cardiac explant tissue taken from dogs post-mortem, with owners’ consent. These cells were able to survive successive passages in serum free media and formed large spherical cell clusters, termed ‘cardiospheres’. CSCs were capable of clonal expansion under controlled culture conditions, demonstrating their ability for self-renewal. Characterisation of these cells demonstrated the expression of CSC markers; c-Kit, GATA 4 and Flk-1 and no expression of cardiac lineage markers including cardiac troponin T and I, Nkx2.5, the cardiac ryanodine receptor and the β1-adrenergic receptor. Primary canine MSCs were isolated from bone marrow aspirates using ficoll separation and cultured on tissue culture plastic. Canine MSCs closely resembled MSCs described from other species, such as the human and mouse, and were found to express CD44 and STRO-1 and were negative for CD34 and CD45. CSCs and MSCs were exposed to published cardiac directed differentiation protocols and differentiation then analysed using cellular morphology and gene expression. Canine CSCs appeared to differentiate partially along cardiac lineages with upregulation of cardiac troponin T and Nkx2.5, and down regulation of c-Kit and endothelial lineage markers. Canine MSCs demonstrated some morphological changes during cardiac differentiation, and demonstrated up-regulation of Nkx2.5 and Flk-1 but no significant alteration in other markers examined. This suggested that cardiac directed differentiation was not as successful with canine MSCs compared to CSCs and conflicting with published data using rodent MSC models. Murine MSCs were used as a positive control cell line for cardiac directed differentiation, based upon published literature. Critically there were key marker expression differences between baseline murine and canine MSCs, including the expression of cardiac markers such as cardiac troponin T and I, and the Ryanodine receptor. Furthermore, expression analysis of cardiac genes changed with time in culture and passage number and no significant alteration was seen when cells were subjected to the cardiac differentiation protocol; thereby bringing into question the data regarding successful cardiac differentiation using murine MSCs. Canine MSCs were further differentiated toward a chondrocyte lineage to investigate the use of MSCs for orthopaedic research. Canine MSCs were successfully differentiated toward articular type cartilage, with demonstration of extracellular matrix secretions, an upregulation of collagen type II with downregulation of collagen type I and the development of SOX9 expression in differentiated cells. This thesis builds the groundwork for future ASC research in the dog. Successful isolation and culture of two ASC sources from the dog is demonstrated. Cardiac and cartilage directed differentiation was successful using primary sourced cells, but differentiation was found to be limited to highly specific routes for each stem cell source. The results presented here highlight the importance of analysing baseline stem cells extensively prior to differentiation and in particular, before making comparisons between cell populations isolated from different locations and species.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Effect of pimobendan on left atrial function in dogs with preclinical myxomatous mitral valve disease

Background: Left atrial (LA) function is an important determinant of left ventricular (LV) filling, playing a key role in maintaining optimal cardiac performance. Pimobendan is a phosphodiesterase III inhibitor with positive inotropic and vasodilator effects. The present study sought to investigate the effects of pimobendan on LA function in dogs with stage B2 myxomatous mitral valve disease (MMVD). Aim: The aim of this investigation was to study the effects of pimobendan on LA function in dogs with preclinical MMVD. Methods: Twenty-seven dogs with stage B2 MMVD were retrospectively included. LA function was assessed prior to and 1–6 months following pimobendan initiation. For each dog, 2-dimensional (2D) echocardiography was performed to assess LA diameter and volume for each phase of the LA cycle and to assess complete, passive and active LA function. Pulsed wave tissue Doppler imaging (TDI) of the left ventricular longitudinal myocardial velocity associated with atrial contraction (A’), both at the level of the interventricular septum and the LV free wall, were also used as an indicator of LA function. Results: There were no significant differences in any of the left atrial variables pre- and post-treatment. Conclusion: Echocardiographic estimates of LA function by 2D diameters and volumes and TDI A’ in dogs with MMVD do not change after treatment with pimobendan