Evaluation of protocols for derivation of cardiac progenitors from human induced pluripotent stem cells
Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2015
Författare
Pernevik, Elin
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Myocardial infarction and heart failure are major causes of death worldwide. The adult heart has limited potential for repair and regeneration of cardiomyocytes damaged by these pathologies and existing drugs and mechanical devices do not always provide long-term solutions. This creates a demand for advanced in vitro models of human cardiac cells that can be used in the search for long term treatments of heart damage. Existing systems commonly used for research include in vitro models using cancerous cell lines, sometimes of non-human origin, and in vivo murine models, raising questions about the translatability of these systems for human cardiac treatments. Ideally human cardiac cells would be used; however, obtaining primary human cells in sufficient numbers for research is very difficult. One solution is use of human induced pluripotent stem cells (iPSC) that theoretically have the potential to differentiate into any cell type of the body and can be expanded to sufficient numbers. Recently cardiac progenitor cells (CPC) were discovered to be present in adult mammalian cardiac tissue at low ratios (Beltrami et al., 2003). CPC have the potential, following specific stimulation, to proliferate and become new cardiomyocytes; therefore providing a novel strategy for the treatment of heart damage. The aim of this project was to evaluate protocols for derivation of CPC from iPSC as this would provide an in vitro model for drug discovery aiming towards cardiac regeneration by small molecule stimulation of CPC already present in the adult heart. Several protocols have already been published for differentiation of stem cells to cardiomyocytes, and some were evaluated for their ability to derive functional CPC from iPSC in comparison to commercial iCell® CPC. The CPC populations during differentiation were characterized with immunocytochemistry (ICC) and real-time quantitative PCR (qPCR) and expression of relevant cardiac progenitor markers PDGFRα, KDR, Isl1, Nkx2.5 among others was detected for the selected Protocol B CPC and cells derived with a commercial CVP medium. Both these CPC populations are bipotent towards the cardiomyocyte and smooth muscle lineages, but an improved endothelial differentiation is required to prove potential multipotency. The Protocol B progenitors also displayed proliferative capacity confirming their progenitor state. There is a need for optimization of robustness and preservation in the progenitor stage, and further comparison to in vivo derived cardiac cells for evaluation of relevance, but these CPC populations show promise for future use as in vitro models of CPC for drug discovery.
Beskrivning
Ämne/nyckelord
Fysik , Grundläggande vetenskaper , Hållbar utveckling , Biologiska vetenskaper , Innovation och entreprenörskap (nyttiggörande) , Physical Sciences , Basic Sciences , Sustainable Development , Biological Sciences , Innovation & Entrepreneurship