Effect of the functional environment on the cardiac differentiation of iPSC

Abstract

Pristine carbon nanotubes (CNTs) have proven to be excellent supports for cardiac cell growth, survival and maturation, as well to improve cellular function, enhance spontaneous beating activity and benefit their cellular structure. Due to the large quantity of cardiomyocytes that have to be replaced for myocardial regeneration, iPSCs are the most promising candidates for robust generation of cardiomyocytes in vivo/vitro. Herein, iPSCs are cultured and differentiated into cardiomyocytes on functionalized carbon nanotubes (fCNTs). For this purpose, a first optimization of the type of plate and the number of iPSCs suitable for the passaging is performed. Thus, 5{middle dot}105 cells per cm2 are cultured in 24-well and 8-well plates. After 19-days of differentiation and maturation, calcium imaging was done to analyze the spontaneous beating behavior by means of beat frequency and amplitude, immunofluorescence was done to observe evaluate the degree of maturation by staining the sarcomere and the cell nucleus. A set of diverse functionalized CNTs were also tested: pristine CNT, ox-CNT, CNT-COOH, CNT-NH2, CNT-NO2 and CNT-SO3. Calcium analysis showed that all but the nitro-functionalization were beating, with acid-and oxygen derivative CNTs producing an increase in frequency with respect to control, while amino-functional groups decrease it. This suggests that the beating and contractile behavior of cardiomyocyte can be modulated according to the cardiac issue to be faced. In addition, CNT-SO3 produces a striated and elongated sarcomere, proper of the real tissue.