Replacement of Mouse Microglia With Human Induced Pluripotent Stem Cell (hiPSC)-Derived Microglia in Mouse Organotypic Slice Cultures.

Microglia, the major immune cells in the brain, are reported to differ in gene expression patterns among species. Therefore, it would be preferable in some cases to use human microglia rather than mouse microglia in microglia-targeted disease research. In the past half a decade, researchers have developed in vivo transplantation methods in which human induced pluripotent stem cell-derived microglia (hiPSC-MG) are transplanted into a living mouse brain. However, in vivo transplantation methods are not necessarily accessible to all researchers due to the difficulty of obtaining the materials needed and the transplantation technique itself. In addition, for in vivo systems for microglia-targeted drug screening, it is difficult to control the pharmacokinetics, especially blood-brain barrier permeability. Therefore, in addition to existing in vivo transplantation systems, the development of an ex vivo transplantation system would help to further evaluate the properties of hiPSC-MG. In this study, we aimed to establish a method to efficiently transplant hiPSC-MG into cultured mouse hippocampal slices. We found that approximately 80% of the total microglia in a cultured slice were replaced by hiPSC-derived microglia when innate microglia were pharmacologically removed prior to transplantation. Furthermore, when neuronal death was induced by applying Kainic acid (KA) to slice cultures, transplanted hiPSC-MG changed their morphology and phagocytosed cell debris. Thus, this study provides a method to transplant hiPSC-MG into the mouse hippocampal slice cultures with a high replacement rate. Because the transplanted microglia survived and exerted phagocytic functions, this method will be useful for evaluating the properties of hiPSC-MG ex vivo.

Extracellular Vesicles Taken up by Astrocytes.

Extracellular vesicles (EVs) are composed of lipid bilayer membranes and contain various molecules, such as mRNA and microRNA (miRNA), that regulate the functions of the recipient cell. Recent studies have reported the importance of EV-mediated intercellular communication in the brain. The brain contains several types of cells, including neurons and glial cells. Among them, astrocytes are the most abundant glial cells in the mammalian brain and play a wide range of roles, from structural maintenance of the brain to regulation of neurotransmission. Furthermore, since astrocytes can take up EVs, it is possible that EVs originating from inside and outside the brain affect astrocyte function, which in turn affects brain function. However, it has not been fully clarified whether the specific targeting mechanism of EVs to astrocytes as recipient cells exists. In recent years, EVs have attracted attention as a cell-targeted therapeutic approach in various organs, and elucidation of the targeting mechanism of EVs to astrocytes may pave the way for new therapies for brain diseases. In this review, we focus on EVs in the brain that affect astrocyte function and discuss the targeting mechanism of EVs to astrocytes.

A live imaging-friendly slice culture method using collagen membranes.

AIM: Organotypic brain slice culture preserves the geographical position of neurons and neuronal circuits. The slice cultures also maintain both non-neuronal cell types and the surrounding extracellular matrix. The interface method has been widely used for slice cultures, in which brain slices are placed on semiporous polytetrafluoroethylene (PTFE) membranes. However, a low optical transparency of PTFE membrane makes it difficult to perform live imaging of deep regions of slice cultures using an inverted microscope. To overcome the issue, we evaluated the suitability of using collagen membranes for slice cultures, especially focusing on live imaging of the cellular dynamics of green fluorescent protein (GFP)-expressing microglia. METHODS: Entorhinohippocampal slices were cultured on either collagen or PTFE membranes. The influence of membrane type on the ability to observe deep regions of slice cultures was examined by live imaging using an inverted microscope. RESULTS: Collagen membranes were thinner and had better optical transparency compared with PTFE membranes. There were no differences in cell viability, density of neurons or microglia. The densify of visible short branches of microglia in live imaging was higher in collagen membranes than PTFE membranes. CONCLUSION: Collagen membranes are suitable for live imaging of cellular dynamics in slice cultures using an inverted microscope.

Vascular Abnormalities and the Role of Vascular Endothelial Growth Factor in the Epileptic Brain.

Epilepsy is a chronic neurological disorder generally defined to be caused by excessive neuronal activity. Thus, excessive neuronal activity is the main target of the currently used antiepileptic drugs (AEDs). However, as many as 30% of epileptic patients show drug resistance to currently available AEDs, which suggests that epilepsy should be attributed not only to neuronal cells but also to other brain cells, such as glial cells and vascular cells. Astrocytes, pericytes, and endothelial cells in particular comprise the blood-brain barrier (BBB), which tightly regulates the exchange of substances between the brain parenchyma and the circulating blood. It has been proposed that BBB dysfunction, especially barrier leakage, exacerbates epileptic progression, and conversely, that epileptic seizures induce barrier leakage. Furthermore, several studies have shown that BBB dysfunction is one of the main causes of drug resistance in epilepsy. To better understand the mechanisms that link BBB dysfunction and intractable epilepsy to gain insights for the future development of treatments, we review and discuss the relationships between epilepsy and brain vascular abnormalities, mainly by focusing on vascular malformation, BBB dysfunction, and excessive angiogenesis. Because these abnormalities have been reported to be caused by vascular endothelial growth factor (VEGF) in the ischemic brain, we discuss the possible role of VEGF in vascular abnormalities in the epileptic brain, in which the upregulation of VEGF levels has been reported. Both glial cells and endothelial cells express VEGF receptors (VEGFRs); thus, these cells are likely affected by increases in VEGF during seizures, which in turn could cause vascular abnormalities. In this review, we review the possible role of VEGF in epilepsy and discuss the mechanisms that link vascular abnormalities and intractable epilepsy.