Human Microglia-Like Cells Differentiated from Monocytes with GM-CSF and IL-34 Show Phagocytosis of α-Synuclein
Andrea Llaves-López1, Elia Micoli1, Carla Belmonte-Mateos1
1Biochemistry and Molecular Biology Unit, Department of Biomedical Sciences, School of Medicine, University of Barcelona, IDIBAPS, Casanova 143, 08036, Barcelona, Catalonia, Spain.
Abstract:
Microglia, the main resident immune cells in the central nervous system, are implicated in the pathogenesis of various neurological disorders. Much of our knowledge on microglial biology was obtained using rodent microglial cultures. To understand the role of microglia in human disease, reliable in vitro models of human microglia are necessary. Monocyte-derived microglia-like cells (MDMi) are a promising approach. This study aimed to characterize MDMi cells generated from adult human monocytes using granulocyte-macrophage colony-stimulating factor and interleukin-34. To this end, 49 independent cultures of MDMI were prepared, and various methodological and functional studies were performed. We show that with this protocol, adult human monocytes develop into microglia-like cells, a coating is unnecessary, and high cell density seeding is preferable. When compared to monocytes, MDMi upregulate the expression of many, but not all, microglial markers, indicating that, although these cells display a microglia-like phenotype, they cannot be considered bona fide human microglia. At the functional level, MDMi phagocytose α-synuclein aggregates and responds to lipopolysaccharide (LPS) by nuclear translocation of the transcription factor nuclear factor-kappaB (NFkappaB) and the upregulation of proinflammatory genes. Finally, a long-lasting silencing of the transcription factor CCAAT/enhancer protein β (C/EBPβ) was achieved by small interfering RNA, resulting in the subsequent downregulation of proinflammatory genes. This supports the hypothesis that C/EBPβ plays a key role in proinflammatory gene program activation in human microglia. Altogether, this study sheds new light on the properties of MDMi cells and supports these cells as a promising in vitro model for studying adult human microglia-like cells.
Insights
Researchers developed a method to create human microglia-like cells (MDMi) from adult monocytes. These cells mimic some microglial functions, offering a valuable in vitro model for studying neurological disorders.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are key immune cells in the central nervous system, crucial for understanding neurological disorders.
- Current research often relies on rodent models, highlighting the need for reliable human in vitro microglia models.
- Monocyte-derived microglia-like cells (MDMi) present a promising avenue for studying human microglia.
Purpose of the Study:
- To characterize adult human monocyte-derived microglia-like cells (MDMi) generated using specific growth factors.
- To assess the methodological requirements and functional capabilities of these MDMi cells.
- To investigate the role of C/EBPβ in the inflammatory response of human microglia.
Main Methods:
- Generated 49 independent cultures of MDMi from adult human monocytes using GM-CSF and IL-34.
- Performed methodological studies on cell culture conditions (e.g., coating, cell density).
- Conducted functional assays including marker expression analysis, phagocytosis of α-synuclein, LPS stimulation, NFκB translocation, and C/EBPβ silencing via siRNA.
Main Results:
- Adult human monocytes differentiated into microglia-like cells under the specified conditions, with high cell density seeding being preferable.
- MDMi cells upregulated many, but not all, microglial markers, exhibiting a microglia-like phenotype but not identical to bona fide microglia.
- Functional studies confirmed MDMi phagocytic activity, inflammatory responses to LPS (NFκB translocation, gene upregulation), and demonstrated that C/EBPβ silencing reduces pro-inflammatory gene expression.
Conclusions:
- The protocol effectively generates adult human microglia-like cells (MDMi) from monocytes.
- MDMi cells represent a valuable in vitro model for studying human microglia, despite not being identical to primary microglia.
- The transcription factor C/EBPβ plays a significant role in activating the pro-inflammatory gene program in human microglia.
