Development and validation of a long-term co-maturation protocol for human stem cell-derived microglia and neuronal networks
5 affiliations
- 1Radboud University Medical Center, Department of Human Genetics, Nijmegen, the Netherlands; Donders Institute for Brain, Cognition and Behaviour, Medical Neuroscience Department, Nijmegen, the Netherlands
- 2Radboud University Medical Center, Department of Human Genetics, Nijmegen, the Netherlands
- 3Department of Molecular Developmental Biology, Radboud Single Cell Center, Radboud University, Nijmegen, the Netherlands
- 4Radboud University Medical Center, Department of Human Genetics, Nijmegen, the Netherlands; Donders Institute for Brain, Cognition and Behaviour, Medical Neuroscience Department, Nijmegen, the Netherlands. Electronic address: [email protected]
- 5Radboud University Medical Center, Department of Human Genetics, Nijmegen, the Netherlands; Donders Institute for Brain, Cognition and Behaviour, Medical Neuroscience Department, Nijmegen, the Netherlands; Radboud University Medical Center, Department of Psychiatry, Nijmegen, the Netherlands. Electronic address: [email protected]
Abstract
Microglia-neuron interactions play a central role in a variety of central nervous system disorders. Technologies using human induced pluripotent stem cells (hiPSCs) have been developed to model human brain cells with the goal to understand their function. To effectively study neuro-immune crosstalk and investigate microglial contributions to neuronal network development and function, both microglia and neurons should co-mature allowing for long-term interactions throughout their differentiation. Here, we present a co-maturation protocol that robustly generates glutamatergic neuronal networks containing human iPSC-derived microglia. We validated the long-term co-cultures using single-cell transcriptomics, imaging and neuronal activity readouts. We show that astrocytes were required for long-term survival of microglia and for their integration into neuronal networks. Our co-maturation approach induced the typical ramified microglia morphology and characteristic microglia-neuron interactions. Homeostatic markers like P2RY12 and TMEM119 and neuronal remodeling associated genes were upregulated compared to microglia monocultures, highlighting the necessity of the environment to generate and maintain the context-dependent microglia signature in vitro. In this manuscript, we include the full optimization process of our co-maturation approach, a comprehensive description of the protocol, practical guidelines and troubleshooting tips. Our co-maturation model provides a powerful tool to assess the role of human microglia in modulating neuronal function and development in health and disease. Highlights ptimized human iPSC differentiation protocol that allows for co-maturation of microglia and Neurogenin2 -induced neuronal networks. are required for long-term survival and integration of microglia into neuronal networks. Co-maturation approach enables characteristic neuron-microglia interactions and induces signature morphology, transcriptome and proteins of human microglia.
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BibTeX
@article{mordelt2026development,
title = {{Development and validation of a long-term co-maturation protocol for human stem cell-derived microglia and neuronal networks}},
author = {Annika Mordelt and Imke M. E. Schuurmans and Nicky Scheefhals and Marina P. Hommersom and Koen Slottje and Kimberly Mast and Mara Graziani and Carlos O. González and Klaas W. Mulder and Laura J. A. Wingens and Dirk Schubert and Nael Nadif Kasri and Lot D. de Witte},
journal = {Stem Cell Reports},
year = {2026},
month = aug,
doi = {10.1016/j.stemcr.2026.103053},
url = {https://doi.org/10.1016/j.stemcr.2026.103053},
note = {CC BY},
}
RIS
TY - JOUR
TI - Development and validation of a long-term co-maturation protocol for human stem cell-derived microglia and neuronal networks
AU - Annika Mordelt
AU - Imke M. E. Schuurmans
AU - Nicky Scheefhals
AU - Marina P. Hommersom
AU - Koen Slottje
AU - Kimberly Mast
AU - Mara Graziani
AU - Carlos O. González
AU - Klaas W. Mulder
AU - Laura J. A. Wingens
AU - Dirk Schubert
AU - Nael Nadif Kasri
AU - Lot D. de Witte
PY - 2026
DA - 2026/08/20
JO - Stem Cell Reports
DO - 10.1016/j.stemcr.2026.103053
UR - https://doi.org/10.1016/j.stemcr.2026.103053
AB - Microglia-neuron interactions play a central role in a variety of central nervous system disorders. Technologies using human induced pluripotent stem cells (hiPSCs) have been developed to model human brain cells with the goal to understand their function. To effectively study neuro-immune crosstalk and investigate microglial contributions to neuronal network development and function, both microglia and neurons should co-mature allowing for long-term interactions throughout their differentiation. Here, we present a co-maturation protocol that robustly generates glutamatergic neuronal networks containing human iPSC-derived microglia. We validated the long-term co-cultures using single-cell transcriptomics, imaging and neuronal activity readouts. We show that astrocytes were required for long-term survival of microglia and for their integration into neuronal networks. Our co-maturation approach induced the typical ramified microglia morphology and characteristic microglia-neuron interactions. Homeostatic markers like P2RY12 and TMEM119 and neuronal remodeling associated genes were upregulated compared to microglia monocultures, highlighting the necessity of the environment to generate and maintain the context-dependent microglia signature in vitro. In this manuscript, we include the full optimization process of our co-maturation approach, a comprehensive description of the protocol, practical guidelines and troubleshooting tips. Our co-maturation model provides a powerful tool to assess the role of human microglia in modulating neuronal function and development in health and disease. Highlights ptimized human iPSC differentiation protocol that allows for co-maturation of microglia and Neurogenin2 -induced neuronal networks. are required for long-term survival and integration of microglia into neuronal networks. Co-maturation approach enables characteristic neuron-microglia interactions and induces signature morphology, transcriptome and proteins of human microglia.
SN - 2213-6711
ER -