Abstract
Gene expression data indicate that during human brain development, neurons change the NMDA receptor (NMDAR) subunit composition to modulate their function, favouring the GluN2A subunit over GluN2B—a hallmark of neuronal maturation. However, evidence supporting this phenomenon in human iPSC-derived neurons remains elusive. Here, using two differentiation methods in parallel (BrainPhys Neuronal Medium, BPM, and Neural Maintenance Medium, NMM), we provide evidence of increased synaptic localization of NMDARs during neuronal maturation and that GluN2A subunit is crucial for the NMDA physiological function-inducing inward currents and calcium entrance at 60 days of differentiation. Calcium responses to specific agonists, particularly NMDA, were elevated in cells cultured under BPM conditions. This is likely attributable to their more mature neuronal phenotype and the RNA-seq identified upregulation of genes involved in intracellular calcium signaling proteins. Our results offer insight into how glutamate receptor subunits mature during brain development, delineating approaches to study NMDAR activity in health and disease.
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Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank Prof. Juan Lerma for generously providing access to his laboratory facilities for the patch clamp experiments, for his assistance with data analysis and for his insightful contributions that greatly enriched this work.
Funding
This work was supported by grants from the Fondo de Investigaciones Sanitarias (PI22/01329, co-funded by the Fondo Europeo de Desarrollo Regional, FEDER “Investing in your future”), CIBERNED (Instituto de Salud Carlos III, Spain), the Instituto de Investigación Sanitaria y Biomédica de Alicante (Isabial), Direcció General de Ciència i Investigació, Generalitat Valenciana (CIAICO/2024/313) and “Severo Ochoa” Program for Centers of Excellence in R&D (CEX2021-001165-S). SE is supported by a PFIS fellowship from the ISC‐III. CAG is supported by a predoctoral contract. (PRE2022-104182) funded by the Agencia Estatal de Investigación (AEI) and the Ministerio de Ciencia e Innovación (MCIN) under the Plan Estatal de I + D+I 2021–2023, co-funded by the European Social Fund (FSE). FS-L is supported by University of Chicago start-up funds. AVP is supported by the Spanish Agency of Research (AEI) under the grants PID2022-136741NB-100 and by the Generalitat Valenciana through the program PROMETEO/2019/014 and CIPROM/2022/08 to Juan Lerma. EDLP and FAP are supported by grant PID2022-140961OB-100 (MCIN/AEI/https://doi.org/10.13039/501100011033), grant PROMETEO/2021/031 (Generalitat Valenciana Government) and “Severo Ochoa” Program for Centers of Excellence in R&D (CEX2021-001165-S). HZ is a Wallenberg Scholar and a Distinguished Professor at the Swedish Research Council supported by grants from the Swedish Research Council (#2023 − 00356, #2022 − 01018 and #2019–02397), the European Union’s Horizon Europe research and innovation programme under grant agreement No 101053962, and Swedish State Support for Clinical Research (#ALFGBG-71320).
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SE and CAG performed the iPSC cultures and differentiation, FACS, ICQ, WB and RT-qPCR and prepared Figs. 1–2. HZ donates the cells and revised the manuscript. MACG participated in the design and culture of iPSC. EDLP and FAP performed the Fura-2 studies and prepared Fig. 4. AVP performed the patch-clamp studies and prepared Fig. 3 and Supp Fig. 2. FSL and SE performed the RNAseq studies and prepared Supp Fig. 1 and Supp Excells. ICI and SE wrote the main manuscript text. ICI and JSV revised it substantively. All authors reviewed the manuscript.
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HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZpath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, Enigma, LabCorp, Merck Sharp & Dohme, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Quanterix, Red Abbey Labs, reMYND, Roche, Samumed, ScandiBio Therapeutics AB, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures sponsored by Alzecure, BioArctic, Biogen, Cellectricon, Fujirebio, LabCorp, Lilly, Novo Nordisk, Oy Medix Biochemica AB, Roche, and WebMD, is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program, and is a shareholder of MicThera (outside submitted work).
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The human cells (iPSCs) were a gift and the original source of the cells, the laboratory of Dr Henrik Zetterberg, confirm that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent. The authors declare that they have not used AI-generated work in this manuscript.
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Escamilla, S., Avilés-Granados, C., Peralta, F.A. et al. GluN2A-mediated currents and calcium signal in human iPSC-derived neurons. Sci Rep (2026). https://doi.org/10.1038/s41598-026-38482-y
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DOI: https://doi.org/10.1038/s41598-026-38482-y
