Development and validation of a simplified method to generate human microglia from pluripotent stem cells

Abstract

BackgroundMicroglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia.ResultsiPSCs are first differentiated toward a mesodermal, hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGFβ-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGFβ signaling (IDE1) can be used to replace recombinant TGFβ1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered.ConclusionWe anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.Electronic supplementary materialThe online version of this article (10.1186/s13024-018-0297-x) contains supplementary material, which is available to authorized users.

Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system

“…This approach reproducibly yields enriched populations of CD43 + CD45 + CD34 + hematopoietic progenitors with functional activity in the colony-forming unit (CFU) assay. The simplicity and efficiency of the protocol have enabled secondary applications, such as differentiation of microglia 25 and NK cells 26 , validation of CD34 fluorescent reporter human iPSC lines 27 , mapping of human pluripotent stem cell differentiation pathways 28 , and investigation of a posttranscriptional regulatory circuitry for the maintenance and differentiation of pluripotent stem cells and HSPCs 29 . In this study, we systematically examined the phenotype and function of supernatant and monolayer cells throughout differentiation, and provide evidence that this system can be adapted to move closer to a clinically-relevant protocol for the generation of definitive erythroid and multipotent hematopoietic progenitor cells.…”

Human iPSC‐derived microglia: A growing toolset to study the brain's innate immune cells

“…Due to the rapid popularity of these models, methods have quickly begun to be refined and even commercialized in an effort to reduce variability and broaden access to these techniques. For example, McQuade and colleagues demonstrated that utilizing a commercial kit to generate CD43+ HPCs greatly simplified the initial paradigm presented by Abud et al (), by eliminating the need for hypoxia and cell sorting (McQuade et al, ). Additionally, this simplified approach led to the production of 60‐fold more HPCs and continued differentiation of these HPCs into iMGs, following the Abud et al () protocol, resulted in iMGs that were transcriptionally and functionally indistinguishable from cells generated with the original approach.…”

“…One of the key benefits to generating microglia from iPSCs is the exponential increase in cell number that can be achieved when compared to the traditional method of isolating microglia from human brain tissue. In general, over 100 million iMGs can be generated from a starting population of just 1 million iPSCs (McQuade et al, ). This improvement allows researchers to perform high throughput experiments that can be well controlled, as the increased cell yield means multiple control groups and technical replicates can be run.…”

“…One of the key benefits to generating microglia from iPSCs is the exponential increase in cell number that can be achieved when compared to the traditional method of isolating microglia from human brain tissue. In general, over 100 million iMGs can be generated from a starting population of just 1 million iPSCs (McQuade et al, 2018).…”

Cell type-specific enhancer-promoter connectivity maps in the human brain and disease risk association

“…Microglia were generated as previously described with minor modifications (66). Flow cytometry was performed on a BD InFlux Cytometer (Becton-Dickinson).…”