Induced pluripotent stem cells regenerative potential in CVD
Research type
Research Study
Full title
Induced pluripotent stem cells regenerative potential in cardiovascular disease
IRAS ID
244958
Contact name
Fiona Lewis
Contact email
Sponsor organisation
Queen Mary University of London
Duration of Study in the UK
2 years, 0 months, 0 days
Research summary
Summary of Research
Cardiovascular disease (CVD) is currently the main cause of death worldwide and at present no widely available restorative treatment option exists. Therefore new strategies are required to identify safe and efficient ways of treating CVD. In recent years, stem cells have emerged as a potentially valuable tool for repair of
damaged hearts and a recent discovery identified that stem cells could be generated from a patient’s own skin cells. To date, these patient-specific cells, termed iPSCs have been generated from different aged patients however evidence suggests that iPSCs generated from ‘aged’ cells have deteriorated functions and may be unsuitable for clinical application. In order to address this we will generate iPSCs from skin cells of ‘aged’ CVD patients and identify their suitability for clinical application. In addition, we will profile secreted nanosized particles, known as exosomes to gain further insight into the implications of reprogramming ‘aged’ cells and uncover their therapeutic potential.Summary of Results
One of the key objectives for this project was to isolate and characterize skin fibroblasts against a panel of age-associated markers. Skin samples from male ischemic heart disease (IHD) donors of different ages (n=10) were collected, skin fibroblasts isolated and expanded in culture before cryopreserving. All isolated skin fibroblasts were validated using an established marker, TE-7 to confirm their identity. Epigenetic screening of human sin fibroblasts revealed a significant correlation between chronological and epigenetic age. Human skin fibroblasts from donors aged 71, 75, 77, and 80 yrs were selected for further analysis including screening against a panel of established ageing markers associated with genomic stability, including 53BP1, Lamin A and telomere length. These were compared to skin fibroblasts isolated from healthy newborn (0 yrs) donors and we identified increased genomic instability associated with increased donor fibroblast age. Together these observations support an accumulation of genomic defects in aged-skin fibroblasts, which we hypothesized may be inherited during reprogramming to induced pluripotent stem cells (iPSCs).To address this key question, we reprogrammed selected skin fibroblasts to iPSCs. We selected both young-healthy and aged-IHD fibroblasts for reprogramming and generated iPSCs using an optimised, non-integrating sendai virus reprogramming system. All successfully reprogrammed iPSCs were validated using a panel of pluripotency markers including, Oct3/4, Nanog, Sox2, SSEA4 and TRA-1-81 using immunocytochemical and flow cytometric analysis. Tri-lineage differentiation of each iPSC line was performed to confirm their pluripotent differentiation potential and all new iPSC lines were karyotyped and matched to their parental fibroblast genomic profile. During this process, we observed a decreased reprogramming efficiency and increased expression of ageing-associated markers in iPSCs from aged-IHD donors as predicted. This suggests that parental fibroblast memory is incompletely erased, aged-IHD iPSCs are more susceptible to damage or a combination of both.
In order to assess the fate of aged-IHD iPSCs upon conversion into cardiomyocytes (CM), we have also spent considerable time refining our iPSC-CM differentiation strategy. Our optimised protocol is now capable of generating beating, cardiac troponin T+ CMs >80% in 15 days. We have confirmed that CMs express cardiac-specific proteins, including Nkx2.5 and cardiac troponin T, with a distinct CM morphology. We have observed significant differences associated with a dysregulated metabolic phenotype in iPSCs reprogrammed from aged-IHD fibroblasts as predicted and we are currently pursuing this line of investigation in more detail to draw meaningful conclusions.
As part of this project, we have also developed a robust protocol for the isolation of secreted extracellular vesicles (EVs) from cell cultures, which express EV markers Alix, TSG101 and CD63 and have a mode particle size <150nm, confirming EV isolations are enriched for small EVs, such as exosomes. We have isolated EVs at different stages of the CM differentiation time course, which has revealed changes in EV abundance and miRNA profile as iPSCs become progressively committed to a CM fate. It remains to be analysed if the number of EVs and/or miRNA cargo are differentially expressed in iPSCs and/or iPSC
REC name
London - Hampstead Research Ethics Committee
REC reference
18/LO/0689
Date of REC Opinion
18 May 2018
REC opinion
Favourable Opinion