Abstract
We characterised the development of spikes, bursts and functional networks in neurons derived from human induced pluripotent stem cells (iPSCs) over a 55-day culture period. Spontaneous neuronal activity was recorded using a multi-electrode array across 20 non-consecutive days. The aim was to track neural maturation by identifying the spatiotemporal patterns of functional connectivity. We identified three distinct activity patterns corresponding to different developmental stages. In the early period (day age 18−23), iPSC-derived neurons exhibited a gradually increasing number of synchronous spikes, firing frequency, network bursts and burst rate, in the mid period (day age 24−28), whereas a fully mature neuronal synchronisation pattern with stable spiking and bursting behaviours were observed, and ultimately iPSC-derived neurons exhibited a decrease in firing rate, number of burst and network bursts for day age 32−55. These patterns exhibited three unique spatial topologies and spectral characteristics: In early period, functional connectivity showed poor neuronal communication with low fluctuation in its temporal dynamics and a 1/f-like spectral distribution. In contrast, a fully mature functional network exhibited strong and broad communication topology, indicating an established iPSC-derived neuronal synchronisation in the mid period, but a decaying network maturation trajectory after day age 32. Staining was also performed on day 21 and day 28 to identify the underlying synaptic mechanisms of functional connectivity obtained from iPSC-derived neuronal population dynamics in MEA. The analysis of Staining showed significant changes in mean volumes of presynaptic and postsynaptic across day 21 and day 28. Our findings suggest that iPSC-derived neurons develop dynamic functional connectivity with distinct communication patterns over time, shedding light on the microscale organisation of neural networks in vitro. This work offers a foundational understanding of in-vitro neuronal network dynamics, which may be valuable for future research into brain function and disorders.
Data availability
The datasets used during the current study can be made available from the corresponding author upon reasonable request and an approval from Monash University Ethics Committee.
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Acknowledgements
The authors thank all members of the lab for their support.
Funding
PK discloses support for the research of this work from a National Health and Medical Research Council (NHMRC) Investigator Grant (GNT2025849). BR discloses support for the research of this work from the Medical Research Future Fund (MRFF) Stem Cell Therapies Mission (MRF1201781).
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S.M. and P.K conceived the experiment. B.R., J.G, M.S.J and A.A.B conducted the experiment, S.M. analysed the data, interpreted the results and wrote the main manuscript. S.M, B.R, T.J.O and P.K edited the manuscript. All authors reviewed the manuscript.
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Mehrkanoon, S., Rollo, B., Gu, J. et al. Distinct functional networks derived from human induced pluripotent stem cell neuronal activity. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40552-0
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DOI: https://doi.org/10.1038/s41598-026-40552-0
