Isolation of cortical mouse oligodendrocyte precursor cells

Abstract

The reliable isolation of primary oligodendrocyte progenitors cells (OPCs) holds promise as both a research tool and putative therapy for the study and treatment of central nervous system (CNS) disease and trauma. Stringently characterized primary mouse OPCs is of additional importance due to the power of transgenics to address mechanism(s) involving single genes. In this study, we developed and characterized a reproducible method for the primary culture of OPCs from postnatal day 5–7 mouse cerebral cortex. We enriched an O4+ OPC population using Magnetic Activated Cell Sorting (MACS) technology. This technique resulted in an average yield of 3.68 × 105 OPCs/brain. Following isolation, OPCs were glial fibrillary acidic protein− (GFAP−) and O4+. Following passage and with expansion, OPCs were O4+, A2B5+, and NG2+. Demonstrating their bi-potentiality, mouse OPCs differentiated into either more complex, highly arborized O4+ or O1+ oligodendrocytes (OLs) or GFAP+ astrocytes. This bi-potentiality is lost, however, in co-culture with rat embryonic day 15 derived dorsal root ganglia (DRG). Following 7–14 days of OPC/DRG co-culture, OPCs aligned with DRG neurites and differentiated into mature OLs as indicated by the presence of O1 and myelin basic protein (MBP) immunostaining. Addition of ciliary neurotrophic factor (CNTF) to conditioned media from OPC/DRG co-cultures improved OPC differentiation into mature O1+ and MBP+ OLs. This method allows for the study of primary mouse cortical OPC survival, maturation, and function without relying on oligosphere formation or the need for extensive passaging.

Bystander mechanism for complement-initiated early oligodendrocyte injury in neuromyelitis optica

“…Oligodendrocyte precursor cell (OPC) cultures from rat brain were generated as described [7, 40]. Briefly, brains from postnatal day 7 pups were harvested and cortex was placed in cold Hank’s balanced salt solution (HBSS, pH 7.2; Invitrogen, Camarillo, CA) without Ca 2+ and Mg 2+ .…”

STAT3 and SOCS3 regulate NG2 cell proliferation and differentiation after contusive spinal cord injury

“…NG2 cells were isolated and cultured as previously described (Dincman et al, 2012) with some modifications. Postnatal day 5–7 mouse brains from either Rosa26-tdTomato F/+ mice, Rosa26-tdTomato F/+ /STAT3 F/F mice, or Rosa26-tdTomato F/+ /SOCS3 F/F mice were manually dissociated into a single cell suspension using a papain based kit (Miltenyi Biotec) according to manufacturer’s instructions.…”

IL4I1 augments CNS remyelination and axonal protection by modulating T cell driven inflammation

“…For CAM and AAM polarization, primary microglia, BV2 cells, and RAW264.7 cells were treated with 100 ng/ml lipopolysaccharide or IL4 for 24 h. Primary oligodendrocyte precursor cell cultures were obtained from postnatal Day 3–5 mouse cortices using magnetic-activated cell sorting (MACS) with anti-O4 microbeads according to the manufacturers protocol (Miltenyi). Once purified, primary oligodendrocyte lineage cells were expanded in growth media (DMEM-F12 with N2, B27, penicillin/streptomycin, bovine serum albumin, PDGF and FGF) for 24 h and then differentiated in defined media (DMEM-F12, N2, B27, penicillin/streptomycin, insulin and T3) without PDGF and FGF for 48 h as previously described (Dincman et al , 2012). In vitro experiments were replicated twice for analysis.…”