Tracking long-term functional connectivity maps in human stem-cell-derived neuronal networks by holographic-optogenetic stimulation

Neuronal networks derived from human induced pluripotent stem cells (hiPSCs) have been exploited widely for modelling neuronal circuits, neurological diseases and drug screening. As these networks require extended culturing periods to functionally mature in vitro, most studies are based on immature networks. To obtain insights on long-term functional features of human networks, we improved a long-term glia-co-culture culturing protocol directly on multi-electrode arrays (MEA), facilitating long-term assessment of electrical features at weekly intervals. We applied optogenetic stimulation to induce neuronal activity, which resulted in accelerated neuronal responses during network development. Using holographic stimulation with single-cell-resolution, propagating evoked activities of 400 individually stimulated neurons per MEA were traceable, and precise network functional connectivity motifs were revealed. Our integrated holographic optogenetic stimulation platform on MEAs facilitates studying long-term functional dynamics of human neuronal networks in vitro. This is an important step towards establishing hiPSC-derived neurons as profound functional testbeds for basic and biomedical research. HighlightsO_LIIntegrated platform allowed long-term optogenetic experiments on hiPSC-derived networks. C_LIO_LIFull-field optogenetic stimulation boosted hiPSC-derived neuronal network activity. C_LIO_LISingle-neuron resolution holographic stimulation evoked local responses in the network. C_LIO_LIHolographic stimulation of each neuron revealed its functional connectivity patterns. C_LIO_LISubsequent holographic stimulation of more than 400 neurons revealed the whole network connectivity map. C_LI