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

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.

SUBMITTER: Washer SJ

PROVIDER: S-EPMC9663826 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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

Washer Sam J SJ Perez-Alcantara Marta M Chen Yixi Y Steer Juliette J James William S WS Trynka Gosia G Bassett Andrew R AR Cowley Sally A SA

Scientific reports 20221114 1

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 ...[more]

PMID: 36376339

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Project description:BackgroundTrans-differentiation of human-induced pluripotent stem cells into neurons via Ngn2-induction (hiPSC-N) has become an efficient system to quickly generate neurons a likely significant advance for disease modeling and in vitro assay development. Recent single-cell interrogation of Ngn2-induced neurons, however, has revealed some similarities to unexpected neuronal lineages. Similarly, a straightforward method to generate hiPSC-derived astrocytes (hiPSC-A) for the study of neuropsychiatric disorders has also been described.ResultsHere, we examine the homogeneity and similarity of hiPSC-N and hiPSC-A to their in vivo counterparts, the impact of different lengths of time post Ngn2 induction on hiPSC-N (15 or 21 days), and the impact of hiPSC-N/hiPSC-A co-culture. Leveraging the wealth of existing public single-cell RNA-seq (scRNA-seq) data in Ngn2-induced neurons and in vivo data from the developing brain, we provide perspectives on the lineage origins and maturation of hiPSC-N and hiPSC-A. While induction protocols in different labs produce consistent cell type profiles, both hiPSC-N and hiPSC-A show significant heterogeneity and similarity to multiple in vivo cell fates, and both more precisely approximate their in vivo counterparts when co-cultured. Gene expression data from the hiPSC-N show enrichment of genes linked to schizophrenia (SZ) and autism spectrum disorders (ASD) as has been previously shown for neural stem cells and neurons. These overrepresentations of disease genes are strongest in our system at early times (day 15) in Ngn2-induction/maturation of neurons, when we also observe the greatest similarity to early in vivo excitatory neurons. We have assembled this new scRNA-seq data along with the public data explored here as an integrated biologist-friendly web-resource for researchers seeking to understand this system more deeply: https://nemoanalytics.org/p?l=DasEtAlNGN2&g=NES .ConclusionsWhile overall we support the use of the investigated cellular models for the study of neuropsychiatric disease, we also identify important limitations. We hope that this work will contribute to understanding and optimizing cellular modeling for complex brain disorders.

Dataset Information

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

Publications

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

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