Human cortical organoids with engineered microglia-like cells [CROP-seq]

Microglia play vital roles in the emergence and preservation of a healthy brain microenvironment with their impaired functions highlighted in neurodevelopmental and neurodegenerative disorders. However, investigating the microglia function in health and disease states has been challenging due to the lack of easily accessible human models. Here, we develop a method to generate functional microglia inside human cortical organoids (hCOs) from human embryonic stem cells (hESCs) and apply this system to dissect the role of microglia under inflammation induced by amyloid- (A). The overexpression of the myeloid-specific transcription factor PU.1 generated microglia-like cells in hCOs, producing mhCOs (microglia-containing hCOs) and engrafted in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement/chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by A. A-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer’s disease (AD) stage III genes was attenuated in mhCOs. Finally, we determined the function of AD-associated genes highly expressed in microglia in response to A by using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. Together, mhCOs represent an innovative platform to investigate neurodegenerative disorders and serve to develop therapeutics in the future. Single-cell CRISPRi Screening of microglia-like cells containing cortical organoids