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

Microglia are central to brain development and homeostasis and are increasingly recognized for their roles in neurodegenerative diseases like Alzheimer’s. Because human and mouse microglia differ significantly, and access to primary human microglia from tissue is limited and often confounded by disease-related activation, researchers have turned to induced pluripotent stem cell (iPSC) models. However, early protocols for generating iPSC-derived microglia were technically complex, requiring expertise in stem cell culture, hypoxic incubation, and expensive cell-sorting techniques like FACS.

The authors designed a simplified protocol (iPS-microglia 2.0) that mimics microglia development. The process starts by differentiating iPSCs into hematopoietic progenitor cells (HPCs) using a commercial kit. These progenitors are then matured into microglia using a specific cocktail of three cytokines: M-CSF, IL-34, and TGFβ-1. This updated method is designed to avoid the hurdles of previous protocols by removing the need for sorting (FACS or MACS), hypoxic chambers, or complex media formulations. To validate the cells, the researchers performed RNA sequencing, assessed phagocytic function against multiple substrates, and confirmed their ability to engraft into the brains of humanized mice.

The study demonstrates that iPS-microglia 2.0 are essentially indistinguishable from microglia produced by more rigorous, previous-generation protocols. Transcriptome analyses show that these cells cluster closely with each other and with human fetal/adult microglia, remaining distinct from other immune cell types like monocytes. Functional tests confirmed that both methods yield cells with identical phagocytic activity against amyloid-beta and other substrates. Additionally, the study explored the use of a small molecule, IDE1, as a cheaper alternative to recombinant TGFβ-1, showing that it can indeed support microglial production, albeit with some transcriptional variation.