Advancing human iPSC-derived motor neuron models using glutamatergic modulators for synaptic function studies

Understanding human synapse structure and function at nanoscale resolution requires experimental systems that support both high-quality imaging and robust synaptogenesis. While brain tissue and primary neuronal cultures have been widely used, human induced pluripotent stem cells (iPSCs) offer key advantages: they enable patient-specific modeling and provide a renewable source of human neurons without relying on animal models. However, reproducible synapse formation in iPSC-derived neurons, particularly motor neurons (MNs), remains challenging due to variable differentiation efficiencies and low synaptic density.

Here, we optimized MN differentiation to enhance synaptogenesis and enable high-resolution structural analysis using cryo-electron tomography (cryo-ET). Within 28 days, human iPSC-derived MNs developed into morphologically and functionally mature neuronal networks, characterized by phase-bright somata, elongated axons, and dense axodendritic branching. To further promote synaptic connectivity, we applied the glutamatergic modulators CX516 and CDPPB during differentiation.

Their effects were validated by immunolabeling of synaptic markers (revealing increased apposition of pre- and postsynaptic proteins), electrophysiology, calcium imaging, live-cell synaptic vesicle recycling assays, and ultrastructural imaging, including conventional EM and cryo-ET, which confirmed the presence of morphologically defined synapses with ultrastructural features consistent with functional synapses. This combined approach of optimized differentiation and targeted neuromodulation yielded reproducible MN networks with enhanced putative synaptic density and function, providing a robust in vitro platform for investigating human MN physiology and synaptic mechanisms, amenable to future disease modeling. We thank the Dubochet Center for Imaging Bern (DCI-Bern) and the Microscopy Imaging Center (MIC) of the University of Bern for their technical support and access to advanced imaging facilities.

We are grateful to Marek Kaminek and David Kalbermatter for their valuable assistance with electron microscopy, and to Beat Haenni and Desirée Dürr for their expert preparation of conventional EM samples. We also thank Diego Haene for his helpful contributions to the experimental work. This work was supported by grants from the Swiss National Science Foundation (31003 A_179520, 32NE30_185536, CRSII-222809 to BZ).

Open access funding provided by University of Bern. These authors contributed equally: Iman Rostami, Jana Leuenberger, Grischa Ott. Institute of Anatomy, University of Bern, Bern, Switzerland Iman Rostami, Jana Leuenberger, Grischa Ott, Amin Khosrozadeh & Benoît Zuber Psychiatrie Biel (Universitäres Psychiatrisches Zentrum Bern), Biel/Bienne, Switzerland Department of Neurology, Inselspital University Hospital, Bern, Switzerland Department of Physical Medicine & Rehabilitation, University of Missouri, Columbia, MO, USA NextGen Precision Health, University of Missouri, Columbia, MO, USA Department of Physiology, University of Bern, Bern, Switzerland The authors declare no competing interests.

The use of human induced pluripotent stem cells (iPSCs) in this study was approved by the Swiss Ethics Committees on research involving humans (swissethics; BASEC project ID 2023-00163) under a protocol covering both ALS patient-derived and healthy control samples. All methods were performed in accordance with the relevant guidelines and regulations. The authors declare their agreement to participate.