Young at Heart: Pioneering Approaches to Model Nonischaemic Cardiomyopathy with Induced Pluripotent Stem Cells

A mere 9 years have passed since the revolutionary report describing the derivation of induced pluripotent stem cells from human fibroblasts and the first in-patient translational use of cells obtained from these stem cells has already been achieved. From the perspectives of clinicians and researchers alike, the promise of induced pluripotent stem cells is alluring if somewhat beguiling. It is now evident that this technology is nascent and many areas for refinement have been identified and need to be considered before induced pluripotent stem cells can be routinely used to stratify, treat and cure patients, and to faithfully model diseases for drug screening purposes. This review specifically addresses the pioneering approaches to improve induced pluripotent stem cell based models of nonischaemic cardiomyopathy

Establishment of a Duchenne muscular dystrophy patient-derived induced pluripotent stem cell line carrying a deletion of exons 51–53 of the dystrophin gene (CCMi003-A)

Abstract Duchenne's muscular dystrophy (DMD) is a neuromuscular disorder affecting skeletal and cardiac muscle function, caused by mutations in the dystrophin (DMD) gene. Dermal fibroblasts, isolated from a DMD patient with a reported deletion of exons 51 to 53 in the DMD gene, were reprogrammed into induced pluripotent stem cells (iPSCs) by electroporation with episomal vectors containing the reprogramming factors: OCT4, SOX2, LIN28, KLF4, and L-MYC. The obtained iPSC line showed iPSC morphology, expression of pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal

Generation of induced pluripotent stem cells from a Becker muscular dystrophy patient carrying a deletion of exons 45-55 of the dystrophin gene (CCMi002BMD-A-9 ∆45-55)

Abstract Becker muscular dystrophy (BMD) is a dystrophinopathy caused by mutations in the dystrophin gene on chromosome Xp21. BMD mutations result in truncated semi-functional dystrophin isoforms. Consequently, less severe clinical symptoms become apparent later in life compared to Duchenne muscular dystrophy. Dermal fibroblasts from a BMD patient were electroporated with episomal plasmids containing reprogramming factors to create the induced pluripotent stem cell line: CCMi002BMD-A-9 that showed pluripotent markers, were karyotypically normal and capable of trilineage differentiation. MLPA analyses performed on DNA extracted from CCMi002BMD-A-9 showed an in-frame deletion of exons 45 to 55 (CCMi002BMD-A-9 Δ45-55)

Derivation of the Duchenne muscular dystrophy patient-derived induced pluripotent stem cell line lacking DMD exons 49 and 50 (CCMi001DMD-A-3, ∆ 49, ∆ 50)

Abstract Duchenne muscular dystrophy (DMD) is caused by abnormalities in the dystrophin gene and is clinically characterised by childhood muscle degeneration and cardiomyopathy. We produced an induced pluripotent stem cell line from a DMD patient's dermal fibroblasts by electroporation with episomal vectors containing: hL-MYC, hLIN28, hSOX2, hKLF4, hOCT3/4. The resultant DMD iPSC line (CCMi001DMD-A-3) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal. MLPA analyses performed on DNA extracted from CCMi001DMD-A-3 showed a deletion of exons 49 and 50 (CCMi001DMD-A-3, ∆ 49, ∆ 50)

An investigation of the molecular signalling events which occur in cannabiniod-mediated neuronal apoptosis

THESIS 8519The plant-derived cannabinoid ?9-Tetrahydrocannabinol, is the predominant psychoactive moiety of cannabis and exerts a variety of psychological and physiological effects in humans. Previous investigations in this laboratory have shown that ?9-Tetrahydrocannabinol (5 ?M) induces apoptosis in cortical neurones via signalling through the cannabinoid receptor type 1. The phosphorylation of the tumour suppressor protein, p53 at serine residue 15 is a critical step in stabilising p53 and promoting p53-induced apoptosis. I report that ?9-Tetrahydrocannabinol activates p53 by inducing the phosphorylation of serine residue 15, which was mediated by the stress activated protein kinase, c-jun N terminal kinase 1. Furthermore, ?9-Tetrahydrocannabinol induced the translocation of phosphorylated-p53 ser15 to the lysosomal membrane; an event that coincided with ?9-Tetrahydrocannabinol-induced lysosomal membrane destabilisation. ?9-Tetrahydrocannabinol also induced the selective translocation of cathepsin-D, a lysosomal protease which was required for ?9- Tetrahydrocannabinol-induced caspase-3 activation and DNA fragmentation. Depleting neurones of p53 using small interfering RNA inhibited ?9-Tetrahydrocannabinol- induced lysosomal membrane destabilisation and DNA fragmentation, indicating that p53 signalling is pivotal in ?9-Tetrahydrocannabinol-induced lysosomal branch of neuronal apoptosis. Additional evidence for the proclivity of ?9-Tetrahydrocannabinol to regulate p53 signalling was demonstrated by the alterations observed in the p53 post translational modifying proteins, murine double minute 2 and small ubiquitin modifier 1. The observed changes in these p53 regulatory proteins could potentially increase the activity of p53, thus promoting ?9-Tetrahydrocannabinol-induced p53-dependent neuronal apoptosis

Multiomic Approaches to Uncover the Complexities of Dystrophin-Associated Cardiomyopathy

Despite major progress in treating skeletal muscle disease associated with dystrophinopathies, cardiomyopathy is emerging as a major cause of death in people carrying dystrophin gene mutations that remain without a targeted cure even with new treatment directions and advances in modelling abilities. The reasons for the stunted progress in ameliorating dystrophin-associated cardiomyopathy (DAC) can be explained by the difficulties in detecting pathophysiological mechanisms which can also be efficiently targeted within the heart in the widest patient population. New perspectives are clearly required to effectively address the unanswered questions concerning the identification of authentic and effectual readouts of DAC occurrence and severity. A potential way forward to achieve further therapy breakthroughs lies in combining multiomic analysis with advanced preclinical precision models. This review presents the fundamental discoveries made using relevant models of DAC and how omics approaches have been incorporated to date

Exploring homophobia and transphobia in primary schools in Ireland

In 2013, the Department of Education and Skills Anti-Bullying Procedures for Primary and Post-Primary Schools were issued, requiring all primary and secondlevel schools to include homophobia and transphobia in their anti-bullying policies and to document and implement prevention and education strategies. This study was conducted in May and June 2015 with parents, teachers and school leaders - 46 people across six schools. It provides new insight into how primary schools are experiencing and approaching the prevention of homophobia and transphobia as well as educating about gender and sexuality identity. The following is a summary of the key findings from across the cohorts of parents, teachers and school leaders. Questions raised and future directions appear at the end of this report