Functional Analysis of a Novel Immortalized Murine Microglia Cell Line in 3D Spheroid Model.

Microglia are the residential immune cells of central nervous system and they are crucial for brain development and homeostasis, as well as the progression of inflammatory brain diseases. To study microglia's physiological and pathological functions, one of the most widely used models is primary microglia culture from neonatal rodents. However, primary microglia culture is time consuming and needs a great number of animals. In our microglia culture, we found a strain of spontaneously immortalized microglia that continued to divide without any known genetic intervention. We confirmed the immortalization of these cells for uninterrupted thirty passages and we named them as immortalized microglia like-1 cells (iMG-1). The iMG-1 cells kept their microglia morphology, and they expressed macrophage/microglia-specific proteins of CD11b, CD68, P2RY12, and IBA1 in vitro. iMG-1 cells were responsive to inflammatory stimulations with lipopolysaccharide (LPS) and Polyinosinic:polycytidylic acid (pIpC), triggering increased mRNA/protein levels of IL1-ß, IL-6, TNF-α, and interferons. LPS and pIpC treated iMG-1 cells also significantly increased their accumulation of lipid droplets (LDs). We also generated a 3D spheroid model using immortalized neural progenitor cells and iMG-1 cells with defined percentages to study neuroinflammation. The iMG-1 cells distributed evenly in spheroids, and they regulated the basal mRNA levels of cytokines of neural progenitors in 3D spheroid. iMG-1 cells were responsive to LPS by increased expression of IL-6 and IL1-ß in spheroids. Together, this study indicated the reliability of iMG-1 which could be readily available to study the physiological and pathological functions of microglia.

The Characterization of a Subependymal Giant Astrocytoma-Like Cell Line from Murine Astrocyte with mTORC1 Hyperactivation.

Tuberous sclerosis complex (TSC) is a genetic disorder caused by inactivating mutations in TSC1 (hamartin) or TSC2 (tuberin), crucial negative regulators of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. TSC affects multiple organs including the brain. The neurologic manifestation is characterized by cortical tubers, subependymal nodules (SEN), and subependymal giant cell astrocytoma (SEGA) in brain. SEGAs may result in hydrocephalus in TSC patients and mTORC1 inhibitors are the current recommended therapy for SEGA. Nevertheless, a major limitation in the research for SEGA is the lack of cell lines or animal models for mechanistic investigations and development of novel therapy. In this study, we generated TSC1-deficient neural cells from spontaneously immortalized mouse astrocytes in an attempt to mimic human SEGA. The TSC1-deficient cells exhibit mTORC1 hyperactivation and characteristics of transition from astrocytes to neural stem/progenitor cell phenotypes. Rapamycin efficiently decreased mTORC1 activity of these TSC1-deficient cells in vitro. In vivo, TSC1-deficient cells could form SEGA-like tumors and Rapamycin treatment decreased tumor growth. Collectively, our study generates a novel SEGA-like cell line that is invaluable for studying mTORC1-driven molecular and pathological alterations in neurologic tissue. These SEGA-like cells also provide opportunities for the development of novel therapeutic strategy for TSC patients with SEGA.