We then tested the functional maturation of hCOs. As early as day 45, hCOs started to show active calcium transient, which was already synchronized and could be blocked by TTX (Figure S5C). We found neuronal disinhibition by bicuculline largely induced synchronization of calcium surges at area-scale, with the effect even stronger than observed in hMGEOs (Figure S5D; Movie S2 and S3; n=2 hCOs). Furthermore, staining with pre-synaptic protein synapsin-1 (SYN1) and post-synaptic protein PSD95 confirmed abundant synaptogenesis within hCOs at this stage (Figure S5E). Together, these results demonstrate that functional neurons were generated and neuronal networks were efficiently established in hMGEOs and hCOs.
