Development of Functional Myocardium for Regenerative Treatment of Heart Disease

James Hudson (2010). Development of Functional Myocardium for Regenerative Treatment of Heart Disease PhD Thesis, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland.

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s4054596_video41_stromalfractionmc.wmv s4054596_video41_stromalfractionmc.wmv Click to show the corresponding preview/stream video/x-ms-wmv 2.42MB 1
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Author James Hudson
Thesis Title Development of Functional Myocardium for Regenerative Treatment of Heart Disease
School, Centre or Institute Australian Institute for Bioengineering and Nanotechnology
Institution The University of Queensland
Publication date 2010-10
Thesis type PhD Thesis
Supervisor Justin Cooper-White
Gary Brooke
Ernst Wolvetang
Total pages 251
Total colour pages 35
Total black and white pages 215
Abstract/Summary Heart failure is initiated by an index event, after which heart function progressively and irreversibly worsens. Current treatments can slow this process, but cannot provide full recovery. The goal of future regenerative treatments is to restore tissue function by replacing damaged tissue with new functional tissue. Cell injections of myoblasts or adult stem cells in Phase II clinical trials have demonstrated modest recoveries in heart function, through paracrine mechanisms. However, they lack the cell retention, the provision of functional cardiomyocytes and the sustained cell survival required for more effective increases in heart function. Furthermore, a simple injection of cardiomyocytes alone, in the absence of a matrix or substrate, is not sufficient for substantial functional heart improvements. This thesis focussed on the development of functional myocardial constructs (MCs) for the potential treatment of heart failure by investigating: • Development of a substrate • Fabrication of MCs using primary cardiomyocytes • Generation of clinically applicable cardiomyocyte source • Improvement of cell expansion using a defined medium A novel substrate, acrylate polypropylene glycol triol (aPPGT), was developed for MC applications. A range of important factors were investigated, including the synthesis, processing, cytotoxicity, biodegradation and mechanical properties. The aPPGT had a tuneable elastic modulus (Ecompressive = 30-500 kPa), thus providing useful substrate for tissue engineering applications. To investigate the biological relevance of aPPGT, human bone marrow-derived multipotent mesenchymal stromal cell (hMSC) behaviour was investigated. Results demonstrated that the growth and differentiation of hMSCs could be modulated by aPPGT substrate elasticity. Together the results indicated that aPPGT is an appropriate substrate for tissue engineering applications. Functional MCs were developed using an aPPGT substrate tailored to the modulus of myocardial tissue (Etensile = 60 kPa). Implantation onto the epicardial surface of mouse hearts for 14 days demonstrated that this substrate can integrate into the heart tissue and that there is a foreign body response that will not prohibit in vivo use of aPPGT substrates. Primary mouse cardiomyocytes were then used to form MCs, however, fabrication of a functional MC also required a stromal cell fraction. Two different cell types were thereafter investigated as potential stromal cell components; mouse heart-derived stromal cell fraction cells and mouse bone-derived multipotent mesenchymal stromal cells (mMSCs). Both stromal cell types facilitated the production of functional MCs, however, each combination gave rise to a construct of different properties. The results demonstrated that mMSCs may provide an alternative and potentially more beneficial stromal fraction for therapeutics because MCs containing these cells display reduced fibrotic extracellular matrix expression, increased connexin 43 expression (gap junction protein for electro-coupling) and increased hepatocyte growth factor expression (angiogenesis inducing factor). Stem/stromal cells were then investigated as potential, clinically applicable, cardiomyocyte cell sources. It was determined that the techniques used in this thesis could not induce early nor late cardiomyocyte differentiation in adult stem/stromal cells including hMSCs, mMSC sub-populations and mouse heart cardiosphere derived cells. Another potential source was therefore investigated; human embryonic stem cells (hESCs). A scaleable 2D cardiac differentiation protocol was developed, which used differentiation into Primitive Streak-like cells using a combination of activin A and bone morphogenic protein-4, followed by cardiac differentiation using an inhibitor of endogenous Wnt production. Using this protocol an efficiency of 5.5 % myosin heavy chain (mature cardiomyocyte marker) positive cells at a density of ~100,000 cells/cm2 was achieved in three different hESC lines. This new method is therefore feasible for generating a human source of cardiomyocytes for MC applications. Controlled expansion of hMSCs was investigated as they may be a potentially useful component of MCs. hMSCs require considerable expansion to achieve clinically relevant cell number for therapeutics and the resulting populations generated are heterogenous, containing stem cells and their progeny. Generally, fetal bovine serum (FBS) is used in the medium for expansion, but has considerable batch-to-batch variability and an unknown composition. In this investigation, FBS was removed from the medium and a serum-free defined medium used. This resulted in the expansion of osteo- chondro- progenitors at the expense of adipo-progenitors, which may not be appropriate for expansion of hMSC populations for MC applications. However, modifications of the defined medium may be used to achieve expansion of particular hMSC sub-populations to provide greater efficacy in treatment of heart disease. This thesis demonstrates that the development of a therapeutic MC requires three equally important components, including a substrate, cardiomyocytes derived from an appropriate cell source and a stromal cell component. Progress has been made in this thesis toward the development of a clinically relevant MC incorporating all these features. However, further investigations are required to optimise and humanise these MCs, which will require both in vitro and in vivo investigations.
Keyword Biomaterials
Cardiac Tissue Engineering
Embroyonic Stem Cell Differentiation
Mesenchymal Stem Cells
Multipotent Mesenchymal Stromal Cells
Cardiac Development
Additional Notes Colour: 30,31,35,52,56,97,98,116,119,138,143,144,147,178,192,194,196, 209,239 Landscape: 44-47,83,84,114,122,152-154,171,175,215 Colour and Landscape: 125,146,149,173,177,190,213,219,221,243-245,247-249 Videos: 8 Video files are also part of the thesis

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Created: Mon, 21 Feb 2011, 18:13:30 EST by Mr James Hudson on behalf of Library - Information Access Service