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Título/Title/Titulo

Electrical stimulus as a key component for maturation and characterization of human induced pluripotent stem cell-derived cardiomyocytes

Introdução/Introduction/Introdución

After an acute myocardial infarction (AMI), cardiomyocytes (CMs) are eliminated and replaced by fibrous tissue with low contractile capacity. The use of human-induced pluripotent stem cells (iPSCs) is a promising strategy to restore cardiac function, since they can differentiate into all cell types from the human body, including CMs (iPSC-CMs). However, the complete characterization of cells generated during differentiation as well as the choice of the optimal cell population to use for cardiac repair need to be further assessed.

Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados

The objectives of this work are: 1) to investigate the effect of electrical stimulus (ES) on the differentiation or maturation process of iPSC-CMs; 2) to evaluate if ES can help to define markers to determine the best iPSC-CMs phenotype to use for therapeutic strategies or disease modeling. iPSCs were reprogrammed from cells isolated from urine and fibroblasts of healthy individuals from the Heart Institute (São Paulo, Brazil). iPSCs were characterized according to morphology, presence of molecular and protein markers, differentiation capacity in the three germinative lines and chromosome integrity. Then, they were differentiated into cardiomyocytes directly in the culture plates (control group) or with ES from day 0 to day 3 of the differentiation (ES group) and were characterized by molecular, cellular and functional parameters. iPSCs clones presented small, juxtaposed, monolayer cells with very well defined margin and expression of pluripotent genes (SOX2, OCT3/4, NANOG and REX 1). Almost all iPSCs population is positive for pluripotency markers such as NANOG (98.74%) and SOX2 (97.71%). Immunofluorescence showed the presence and correct localization of pluripotency nuclear (SOX2, NANOG, OCT3/4) and membrane markers (SSEA-4, TRA-1-60, TRA-1-81). 1 iPSC clone formed teratoma when injected in imunodeficient mice and through histological analysis it was possible to verify the presence of the 3 germ layers. The 3 iPSCs clones that presented normal karyotype were success differentiated into cardiomyocytes and spontaneously beating iPSC-CMs were observed at 8 to 10 days after beginning of differentiation. iPSC-CMs with ES presented contractile cells 71% of the time, while control iPSC-CMs only 57%. The percentage of positive cells for cardiac progenitor (NKX2-5) was increased for ES group at day 6 of differentiation, while GATA4 and troponin positive cells were higher at day 15. Moreover, ES group showed higher expression of GATA4 and cardiac genes (TNNI3, CALM3 and MYL2) at day 15 as examined by qPCR. Regarding functional parameters, typical calcium (Ca) handling parameters, such as Ca transient amplitude, time to peak and Ca decay rate were significantly higher in iPSC-CMs with ES compared to the control.

Considerações Finais/Final considerations/Consideraciones finales

In conclusion, these results show that ES can enhance cardiac differentiation efficiency of iPSCs, upregulate cardiac genes and promote functional CMs maturation. This study may contribute to increase the efficiency of the differentiation process of hiPSCs into CMs, and to identify best markers for testing therapeutic iPSC-CMs (after AMI, for example) or disease modeling.

Palavras-chave/Key words/Palabras clave

human-induced pluripotent stem cells, cardiac differentiation, cardiomyocytes, electrical stimulus