Different Contacted Cell Types Contribute to Acquiring Different Properties in Brain Microglial Cells upon Intercellular Interaction

Abstract

Microglial cells (MGs), originally derived from progenitor cells in a yolk sac during early development, are glial cells located in a physiological and pathological brain. Since the brain contains various cell types, MGs could frequently interact with different cells, such as astrocytes (ACs), pericytes (PCs), and endothelial cells (ECs). However, how microglial traits are regulated via cell–cell interactions by ACs, PCs, or ECs and how they are different depending on the contacted cell types is unclear. This study aimed to clarify these questions by coculturing MGs with ACs, PCs, or ECs using mouse brain-derived cells, and microglial phenotypic changes were investigated under culture conditions that enabled direct cell–cell contact. Our results showed that ACs or PCs dose-dependently increased the number of MG, while ECs decreased it. Microarray and gene ontology analysis showed that cell fate-related genes (e.g., cell cycle, proliferation, growth, death, and apoptosis) of MGs were altered after a cell–cell contact with ACs, PCs, and ECs. Notably, microarray analysis showed that several genes, such as gap junction protein alpha 1 (Gja1), were prominently upregulated in MGs after coincubation with ACs, PCs, or ECs, regardless of cell types. Similarly, immunohistochemistry showed that an increased Gja1 expression was observed in MGs after coincubation with ACs, PCs, or ECs. Immunofluorescent and fluorescence-activated cell sorting analysis also showed that calcein-AM was transferred into MGs after coincubation with ACs, PCs, or ECs, confirming that intercellular interactions occurred between these cells. However, while Gja1 inhibition reduced the number of MGs after coincubation with ACs and PCs, this was increased after coincubation with ECs; this indicates that ACs and PCs positively regulate microglial numbers via Gja1, while ECs decrease it. Results show that ACs, PCs, or ECs exert both common and specific cell type-dependent effects on MGs through intercellular interactions. These findings also suggest that brain microglial phenotypes are different depending on their surrounding cell types, such as ACs, PCs, or ECs.

Transplantation of Human Brain-Derived Ischemia-Induced Multipotent Stem Cells Ameliorates Neurological Dysfunction in Mice After Stroke

“…h-iSC-derived cell clusters were fixed and examined by electron microscope (EM) as described. 18 , 23 , 24 …”

Microglia Negatively Regulate the Proliferation and Neuronal Differentiation of Neural Stem/Progenitor Cells Isolated from Poststroke Mouse Brains

“…At the time of tissue harvest, mice were intraperitoneally administered a mixture containing medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) [ 2 , 6 ], followed by transcardial perfusion with 4% paraformaldehyde [ 1 , 3 , 10 ]. The brains were removed and placed in 4% paraformaldehyde for 24 h, followed by incubation in 30% sucrose for cryoprotection at −80 °C.…”

“…Positive areas for nestin and Iba-1 were evaluated in ischemic and peri-ischemic areas in coronal brain sections obtained from the same region across all animals. A total of 27 data points (3 areas/section and 3 sections/brain, 3 animals/each day) using ImageJ, as previously described [ 1 , 6 ]. Ischemic and peri-ischemic areas were defined as regions within the borders of stroke-affected areas and regions within a diameter of 100 µm around the stroke-affected areas, respectively, as described [ 1 , 6 ].…”

“…Regionally-derived endogenous iNSPCs were isolated from ischemic areas of mice after MCAO and maintained in Dulbecco’s modified Eagle’s medium/F12 (Thermo Fisher Scientific, Waltham, MA, USA) containing 20 ng/mL basic fibroblast growth factor (bFGF; Peprotech, Rocky Hill, NJ, USA), 20 ng/mL epidermal growth factor (EGF; Peprotech), 1% N2 (Thermo Fisher Scientific), and 2% fetal bovine serum (FBS), as previously described [ 11 ]. Commercially available mouse brain MGs/MΦs (#SCC134; EMD Millipore, Temecula, CA, USA), ACs (#M1800; ScienCell Research Laboratories, Carlsbad, CA, USA), PCs (#M1200; ScienCell Research Laboratories), and ECs (CRL-2299; ATCC, Manassas, VA, USA) were maintained in appropriate media in accordance with the manufacturer’s protocols, as described [ 6 ]. In some experiments, iNSPCs transfected with GFP-expressing lentivirus vectors and MGs/MΦs, ACs, ECs, and PCs transfected with mCherry-expressing lentivirus vectors were used [ 6 , 10 , 12 ].…”

“…In addition to iNSPC induction, inflammatory cells such as microglial cells (MGs) and macrophages (MΦs), which play an important role in tissue repair following various types of brain injury [ 4 , 5 ], emerge within and around the affected sites. Although their precise role in pathologic processes underlying ischemic injury remains unclear, MGs/MΦs gradually accumulate within and around the injured areas and finally occupy these regions after an ischemic stroke [ 6 ]. In contrast, we previously demonstrated that the number of iNSPCs in areas affected by an ischemic stroke declined at later time points following injury [ 2 , 3 ].…”