A novel chimeric brain slice culture model to study human microglia in homeostasis and disease

Abstract

Microglia are the resident immune cells in the central nervous system (CNS) and as such of high importance for brain homeostasis. Microglial contribution to neurodegenerative diseases has only been fully recognized in the last decade, and our understanding of their role in disease pathogenesis remains incomplete. Recent studies revealed significant differences between murine and human microglia in terms of their gene expression profiles, morphology and responses to pathology. Furthermore, it has become evident that the brain environment is crucial for understanding microglial homeostasis. In this thesis, I describe the establishment of a novel model system to study human induced pluripotent stem cell-derived microglia cells (iMic) in ex vivo brain tissue. To generate chimeric brain slice cultures (cBSC), microglia in murine organotypic hippocampal slice cultures were depleted and replaced by human iMic precursor cells. iMics in cBSCs diferentiated and matured to closely resemble human ex vivo microglia in their morphology, network parameters and transcriptome. Furthermore, iMics responded to focal laser lesions and global immunogenic stimulation with shielding of the lesion site and release of cytokines, respectively, and supported neuronal activity for several months. Interestingly, cBSCs sustained human microglia without the supplementation of human colony stimulating factor 1 (CSF1) and interleukin-34 (IL34), contrasting in vivo xenotransplantation models. Using in vitro, cell-free and in silico approaches, I could demonstrate a cross-species interaction between human CSF1-receptor and its cognate murine ligands CSF1 and IL34. The transplantation of CSF1R loss-of-function iMics verified that CSF1R signaling was required for survival and differentiation, whereas in silico modeling of receptor:ligand interactions and the analysis of binding kinetics pointed to murine IL34 as the primary interaction partner. The latter was confirmed by blocking murine IL34 in cBSCs and by cytokine stimulation assays. Finally, cBSCs were adopted to model disease conditions. The induction of a-synuclein (aSyn) pathology in cBSCs resulted in wide-spread neuronal aSyn lesions and intra-microglial inclusions of aggregated proteins, as observed in murine microglia. Additionally, aSyn-treated iMics presented transcriptional phenotypes described in human Alzheimer’s and Parkinson’s disease patients, such as the upregulation of disease-associated microglia genes including APOE and TREM2. These observations emphasize the opportunities cBSCs offer for studying microglia in disease conditions. In conclusion, cBSCs are a promising new tool to study human iPSC-derived microglia in a biologically relevant environment under both homeostatic and disease conditions. This model can be easily adapted for screening of potential therapeutics or for delineating the cell type- specific effect of disease-associated mutations by utilizing brain slice cultures from genetically engineered mouse models and by using mutant iPSC lines.