Two methods for three - dimensional culture: addition of exogenous ECM components and capture of endogenous ECM components

The culture of cells in hydrogels and scaffolds that mimic in vitro tissue has become increasing popular now there is an understanding of how important the role of ECM is in supporting and directing the behaviour of cells. As such there is a wide range of culture methods varying in complexity, that are available, each of which varies in complexity. From synthetic hydrogels with tuneable mechanical properties or natural (polysaccharide or protein) hydrogels that allow for natural interactions with ECM components, that more accurately represent the compisition of tissues and cells. This thesis looks at two different models of hydrogels, with opposite approaches. First, was an adipogenic model that used a top-down approach, where a native ECM protein (collagen) was used to form a natural hydrogel. Research into adipogenesis is important as obesity becomes more prevalent. Models that represent adipogenesis are important to combat the rise in obesity as they can become high content screening models for anti-obesity drugs. This model used an alginate fibre surrounding a collagen I core with mouse mesenchymal stem cells. Over the course of two weeks, this was found to be a viable model, that supported adipogenesis of mesenchymal stem cells. The creation of the fibre was simple, but requires optimisation to prevent alginate from blocking equipment.. The second model was developed using a bottom-up approach where synthetic self-assembling peptides were selected for the presence of a charge and then used to make a hydrogel. The peptide amphiphiles were designed to have increasing lysine residues (K2, K3 and K4) or a negative glutamic acid residue for a PA with a negative charge (E3). The presence of a charge was theorised to sequester native ECM components. Human induced pluripotent stem cells were in clusters on top of the range of PA hydrogels, a neutral hydrogel (FEFEFKFK) and Matrigel. This synthetic PA hydrogels (K2 and K3) sequestered a glycosaminoglycan secreted by human induced pluripotent stem cells. There was a change in cellular behaviour when cultured on hydrogels with different charges, there was increased attachment to the hydrogel on the negative PA (E3) hydrogel, than all others except Matrigel. There was also increased proliferation on the most positive PA (K4) hydrogel than all other hydrogels except Matrigel. Together this suggested that differently charged hydrogels may be able to create different ECM environments through the sequestration of natively secreted ECM components. The charged PA hydrogels have the potential to form a complex ECM with a large range of ECM components, that cannot be replicated by human design. In addition, the synthesised ECM components will be able to be modified by the cells throughout their lifetime, thereby replicating natural processes. This model could therefore be used to produce representative models for use in a wide range of systems many applications, for example drug testing, disease research and tissue regeneration

Two methods for three - dimensional culture: addition of exogenous ECM components and capture of endogenous ECM components

The culture of cells in hydrogels and scaffolds that mimic in vitro tissue has become increasing popular now there is an understanding of how important the role of ECM is in supporting and directing the behaviour of cells. As such there is a wide range of culture methods varying in complexity, that are available, each of which varies in complexity. From synthetic hydrogels with tuneable mechanical properties or natural (polysaccharide or protein) hydrogels that allow for natural interactions with ECM components, that more accurately represent the compisition of tissues and cells. This thesis looks at two different models of hydrogels, with opposite approaches. First, was an adipogenic model that used a top-down approach, where a native ECM protein (collagen) was used to form a natural hydrogel. Research into adipogenesis is important as obesity becomes more prevalent. Models that represent adipogenesis are important to combat the rise in obesity as they can become high content screening models for anti-obesity drugs. This model used an alginate fibre surrounding a collagen I core with mouse mesenchymal stem cells. Over the course of two weeks, this was found to be a viable model, that supported adipogenesis of mesenchymal stem cells. The creation of the fibre was simple, but requires optimisation to prevent alginate from blocking equipment.. The second model was developed using a bottom-up approach where synthetic self-assembling peptides were selected for the presence of a charge and then used to make a hydrogel. The peptide amphiphiles were designed to have increasing lysine residues (K2, K3 and K4) or a negative glutamic acid residue for a PA with a negative charge (E3). The presence of a charge was theorised to sequester native ECM components. Human induced pluripotent stem cells were in clusters on top of the range of PA hydrogels, a neutral hydrogel (FEFEFKFK) and Matrigel. This synthetic PA hydrogels (K2 and K3) sequestered a glycosaminoglycan secreted by human induced pluripotent stem cells. There was a change in cellular behaviour when cultured on hydrogels with different charges, there was increased attachment to the hydrogel on the negative PA (E3) hydrogel, than all others except Matrigel. There was also increased proliferation on the most positive PA (K4) hydrogel than all other hydrogels except Matrigel. Together this suggested that differently charged hydrogels may be able to create different ECM environments through the sequestration of natively secreted ECM components. The charged PA hydrogels have the potential to form a complex ECM with a large range of ECM components, that cannot be replicated by human design. In addition, the synthesised ECM components will be able to be modified by the cells throughout their lifetime, thereby replicating natural processes. This model could therefore be used to produce representative models for use in a wide range of systems many applications, for example drug testing, disease research and tissue regeneration

Live Simultaneous Monitoring of Mineral Deposition and Lipid Accumulation in Differentiating Stem Cells

Mesenchymal stem cells (MSCs) are progenitors for bone-forming osteoblasts and lipid-storing adipocytes, two major lineages co-existing in bone marrow. When isolated in vitro, these stem cells recapitulate osteoblast or adipocyte formation if treated with specialised media, modelling how these lineages interact in vivo. Osteogenic differentiation is characterised by mineral deposits accumulating in the extracellular matrix, typically assessed using histological techniques. Adipogenesis occurs with accumulation of intracellular lipids that can be routinely visualised by Oil Red O staining. In both cases, staining requires cell fixation and is thus limited to end-point assessments. Here, a vital staining approach was developed to simultaneously detect mineral deposits and lipid droplets in differentiating cultures. Stem cells induced to differentiate produced mixed cultures containing adipocytes and bone-like nodules, and after two weeks live cultures were incubated with tetracycline hydrochloride and Bodipy to label mineral- and lipid-containing structures, respectively. Fluorescence microscopy showed the simultaneous visualisation of mineralised areas and lipid-filled adipocytes in live cultures. Combined with the nuclear stain Hoechst 33258, this approach further enabled live confocal imaging of adipogenic cells interspersed within the mineralised matrix. This multiplex labelling was repeated at subsequent time-points, demonstrating the potential of this new approach for the real-time high-precision imaging of live stem cells

Atrial fibrillation and risk of progressive heart failure in patients with preserved ejection fraction heart failure

Abstract Aims Understanding of the pathophysiology of progressive heart failure (HF) in patients with heart failure with preserved ejection fraction (HFpEF) is incomplete. We sought to identify factors differentially associated with risk of progressive HF death and hospitalization in patients with HFpEF compared with patients with HF and reduced ejection fraction (HFrEF). Methods and results Prospective cohort study of patients newly referred to secondary care with suspicion of HF, based on symptoms and signs of HF and elevated natriuretic peptides (NP), followed up for a minimum of 6 years. HFpEF and HFrEF were diagnosed according to the 2016 European Society of Cardiology guidelines. Of 960 patients referred, 467 had HFpEF (49%), 311 had HFrEF (32%), and 182 (19%) had neither. Atrial fibrillation (AF) was found in 37% of patients with HFpEF and 34% with HFrEF. During 6 years follow‐up, 19% of HFrEF and 14% of HFpEF patients were hospitalized or died due to progressive HF, hazard ratio (HR) 0.67 (95% CI: 0.47–0.96; P = 0.028). AF was the only marker that was differentially associated with progressive HF death or hospitalization in patients with HFpEF HR 2.58 (95% CI: 1.59–4.21; P < 0.001) versus HFrEF HR 1.11 (95% CI: 0.65–1.89; P = 0.7). Conclusions De novo patients diagnosed with HFrEF have greater risk of death or hospitalization due to progressive HF than patients with HFpEF. AF is associated with increased risk of progressive HF death or hospitalization in HFpEF but not HFrEF, raising the intriguing possibility that this may be a novel therapeutic target in this growing population