Validation of hiPSC-NKX2.5-GFP line for cardiac differentiation

• Autor(a) principal: Santos, Fernando Augusto Gonçalves dos
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10348/10586
• topic: cardiomyocytes; cardiovascular diseases

The induced pluripotent stem cell (iPSC) technology has created great expectations to advance the development of therapies for cardiovascular diseases, the leading cause of death worldwide. Accordingly, human iPSCs (hiPSCs) efficiently differentiate to cardiomyocytes (hiPSC-CM) which has been already applied in pre-clinical studies, among others, to improve heart regeneration after myocardial infarction. Nevertheless, it is suggested that therapeutic efficiency of hiPSC-CM may be related to the stage of their development. Thus, the main aim of this study was to validate the hiPSC-NKX2.5-GFP line, that enables expression of a cardiacspecific transcription factor, NKX2.5 which is also an established marker of cardiac progenitor cells, and a reporter gene, green fluorescent protein (GFP) from the same promoter, allowing the identification and facilitating the isolation of early cardiomyocytes in the first days of hiPSCs differentiation. Using polymerase chain reaction (PCR) we confirmed the insertion of the GFP-encoding sequence into the locus of the NKX2.5 gene. hiPSCs were subjected to cardiac differentiation. Flow cytometric analysis revealed the presence of cardiac troponin T- and GFP-positive cells among differentiating hiPSCs. The sorted population of GFP-positive cells also confirmed the insertion of the GFP-encoding sequence identified through reverse transcription PCR. The results proved the possibility of the detection of cardiac progenitor cells based on GFP fluorescence dependent on NKX2.5 activation. A quantitative measurement of the differentiation efficiency confirmed that the method used for the genetic construction of tested cell line does not interfere with its cardiac differentiation potential. Therefore, it could be concluded that the hiPSC-NKX2.5-GFP line can be used for identification and isolation of early cardiomyocytes being a valuable tool for studies of therapeutic approaches to treat cardiovascular diseases.