Development of the central nervous system is characterized by the occurrence of spontaneous synchronized neuronal activity8. To study cortical network connectivity, synchrony, and function, mostly rodent brain slices and primary cortical neuronal cell cultures are used. For example, ex vivo population synchrony was found in hippocampal and cortical slices56, and synchronized bursts were recapitulated using dissociated primary neuronal cultures in vitro17. More recently, hiPSC-derived cortical neurons have been shown to display bursts of synchronized network activity as well, within a specified time span of differentiation30. We were not able to reproduce these data, which might be due to subtle differences in the differentiation protocol, usage of different hiPSC lines, reprogramming methods or methods to measure spontaneous calcium activity. When co-culturing our hiPSC-derived neurons with primary human astrocytes in the presence of the Notch signaling inhibitor DAPT the most reproducible and sustained synchronized network activity was achieved, starting three weeks after final plating of neurons (~DIV50) and sustainable up to 8 weeks (~DIV90) in vitro, with neglectable variation between two different hiPSC lines. The support of primary human astrocytes, via released
