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Single-cell transcriptomics defines an improved, validated monoculture protocol for differentiation of human iPSCs to microglia

View ORCID ProfileSam J Washer, Marta Perez-Alcantara, Yixi Chen, Juliette Steer, William S James, Andrew R Bassett, Sally A Cowley
doi: https://doi.org/10.1101/2022.08.02.502447
Sam J Washer
1James and Lillian Martin Centre for Stem Cell Research, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE
2Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA
3Open Targets, Wellcome Genome Campus, Hinxton, CB10 1SA
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  • ORCID record for Sam J Washer
  • For correspondence: sam.washer@path.ox.ac.uk sally.cowley@path.ox.ac.uk
Marta Perez-Alcantara
2Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA
3Open Targets, Wellcome Genome Campus, Hinxton, CB10 1SA
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Yixi Chen
2Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA
3Open Targets, Wellcome Genome Campus, Hinxton, CB10 1SA
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Juliette Steer
2Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA
3Open Targets, Wellcome Genome Campus, Hinxton, CB10 1SA
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William S James
1James and Lillian Martin Centre for Stem Cell Research, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE
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Andrew R Bassett
2Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA
3Open Targets, Wellcome Genome Campus, Hinxton, CB10 1SA
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Sally A Cowley
1James and Lillian Martin Centre for Stem Cell Research, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE
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  • For correspondence: sam.washer@path.ox.ac.uk sally.cowley@path.ox.ac.uk
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Abstract

There is increasing genetic evidence for the role of microglia in neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and motor neuron disease. Therefore, there is a need to generate authentic in vitro models to study human microglial physiology. Various methods have been developed using human induced Pluripotent Stem Cells (iPSC) to generate microglia, however, systematic approaches to identify which media components are actually essential for functional microglia are mostly lacking. Here, we systematically assess medium components, coatings, and growth factors required for iPSC differentiation to microglia. Using single-cell RNA sequencing, qPCR, and functional assays, with validation across two labs, we have identified several medium components from previous protocols that are redundant and do not contribute to microglial identity. We provide an optimised, defined medium which produces both transcriptionally and functionally relevant microglia for modelling microglial physiology in neuroinflammation and for drug discovery.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted August 03, 2022.
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Single-cell transcriptomics defines an improved, validated monoculture protocol for differentiation of human iPSCs to microglia
Sam J Washer, Marta Perez-Alcantara, Yixi Chen, Juliette Steer, William S James, Andrew R Bassett, Sally A Cowley
bioRxiv 2022.08.02.502447; doi: https://doi.org/10.1101/2022.08.02.502447
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Single-cell transcriptomics defines an improved, validated monoculture protocol for differentiation of human iPSCs to microglia
Sam J Washer, Marta Perez-Alcantara, Yixi Chen, Juliette Steer, William S James, Andrew R Bassett, Sally A Cowley
bioRxiv 2022.08.02.502447; doi: https://doi.org/10.1101/2022.08.02.502447

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