Stem-cell-derived human microglia transplanted into mouse brain to study human disease

Abstract

Microglia are critically involved in complex neurological disorders with a strong genetic component, such as Alzheimer’s disease, Parkinson’s disease and frontotemporal dementia. Although mouse microglia can recapitulate aspects of human microglia physiology, they do not fully capture the human genetic aspects of disease and do not reproduce all human cell states. Primary cultures of human microglia or microglia derived from human induced pluripotent stem cells (PSCs) are difficult to maintain in brain-relevant cell states in vitro. Here we describe MIGRATE (microglia in vitro generation refined for advanced transplantation experiments, which provides a combined in vitro differentiation and in vivo xenotransplantation protocol to study human microglia in the context of the mouse brain. This article details an accurate, step-by-step workflow that includes in vitro microglia differentiation from human PSCs, transplantation into the mouse brain and quantitative analysis of engraftment. Compared to current differentiation and xenotransplantation protocols, we present an optimized, faster and more efficient approach that yields up to 80% chimerism. To quantitatively assess engraftment efficiency by flow cytometry, access to specialized flow cytometry is required. Alternatively, the percentage of chimerism can be estimated by standard immunohistochemical analysis. The MIGRATE protocol takes ~40 d to complete, from culturing PSCs to engraftment efficiency assessment.

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Fig. 1: MIGRATE protocol schematic.
Fig. 2: Schematic to show an overview of the MIGRATE protocol.
Fig. 3: Xenotransplantation schematic.
Fig. 4: In vivo engraftment efficiency is reproducible across different ESC/iPSC cell lines when using the MIGRATE protocol.
Fig. 5: MIGRATE microglial progenitor xenotransplantation reaches high chimerism.

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Acknowledgements

This project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (grant agreement no. ERC-834682 CELLPHASE_AD). This work was also supported by UK Dementia Research, the Flanders Institute for Biotechnology (VIB vzw), Vlaams Initiatief voor Netwerken voor Dementie Onderzoek (Strategic Basic Research Grant no. 135043), a Methusalem grant from KU Leuven and the Flemish Government, the Fonds voor Wetenschappelijk Onderzoek, KU Leuven, The Queen Elisabeth Medical Foundation for Neurosciences, the Opening the Future campaign of the Leuven Universitair Fonds, the Belgian Alzheimer Research Foundation and the Alzheimer’s Association USA. R.M. has funding from Fonds voor Wetenschappelijk Onderzoek (grant no. G0C9219N) and is a recipient of a postdoctoral fellowship from the Alzheimer’s Association USA (fellowship no. 2018-AARF-591110). B.D.S. holds the Bax-Vanluffelen Chair for Alzheimer’s Disease. B.D.S. receives funding from the Medical Research Council, the Alzheimer’s Society and Alzheimer’s Research UK via the Dementia Research Institute. N.F. is recipient of a PhD fellowship from Fonds voor Wetenschappelijk Onderzoek (fellowship no. 1139520N). A.M.-M. is supported by a fellowship from the Alzheimer’s Association USA (fellowship no. AARF-20-684397) and a Marie Skłodowska-Curie Actions - Seal of Excellence Postdoctoral Fellowship. We thank V. Hendrickx and J. Verwaeren for animal husbandry. Imaging was acquired through Nikon A1R Eclipse Ti confocal from LiMoNe facility at CBD. We thank S. Balusu and J. Van Den Daele for providing stem cell expertise.

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Contributions

N.F., A.M.-M., B.D.S. and R.M. conceived the study. N.F., A.M.-M. and R.M. performed all the experiments and wrote the manuscript. I.G. and L.W. assisted with microglia xenotransplantations and engraftment efficiency analysis. All authors read and approved the final manuscript for publication.

Corresponding authors

Correspondence to Bart De Strooper or Renzo Mancuso.

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Competing interests

The authors declare no competing interests. B.D.S. receives grants from different companies that support his research and is a consultant for several companies, but nothing is directly related to the current publication.

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Peer review information Nature Protocols thanks Valentina Fossati and Abed Alfattah Mansour for their contribution to the peer review of this work.

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Key reference using this protocol

Mancuso, R. et al. Nat. Neurosci. 22, 2111–2116 (2019): https://doi.org/10.1038/s41593-019-0525-x

Extended data

Extended Data Fig. 1 MIGRATE protocol representative pictures.

Representative day-by-day pictures of the MIGRATE differentiation protocol (H9 cells). The plates used at the two main stages are reported: days 0 to 4 are performed in low-adherent U-form 96-well plates; days 4 to 18 are performed in regular six-well plates. Media changes are indicated in the appropriate pictures with cytokines acronyms (see ‘Procedure’). Length of scale bars is indicated in the images (10 μm for days 15, 17 and 18; 100 μm for all other pictures).

Supplementary information

Supplementary Information

Supplementary Methods (histological analysis), Supplementary Tables 1 and 2 and Supplementary Fig. 1.

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Fattorelli, N., Martinez-Muriana, A., Wolfs, L. et al. Stem-cell-derived human microglia transplanted into mouse brain to study human disease. Nat Protoc (2021). https://doi.org/10.1038/s41596-020-00447-4

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