Characteristics of oligodendroglial lineage progression in a novel, non-inducible neuron-tri-glial coculture system

The brain mainly consists of four mature cell types—neurons, astrocytes, microglia, and oligodendrocytes—that work together to maintain brain homeostasis. However, the primary neuron-astrocyte-microglia tri-culture lacked oligodendrocyte lineage cells, including oligodendrocyte precursor cells (OPCs), immature (or pre-myelinating) oligodendrocytes, and mature (or myelinating) OLs, which form myelin sheaths that wrap neuronal axons and facilitate rapid action potential propagation. This study aimed to establish a high-throughput, non-inducible neuron-tri-glial coculture system incorporating the main brain cell types from embryonic day 14 brains, with optimized cell density and incubation under different nutritional or inflammatory conditions, using multiple approaches, including immunofluorescence staining, Western blotting, single-cell RNA sequencing, and ELISA. Our data showed that, with optimized cell density (2 × 10 5 cells/well) in 96-well plates and a time course from 14 to 21 days after cell seeding, oligodendrocyte lineage progression from oligodendrocyte progenitor cells to mature oligodendrocytes occurred in non-inducible neuron-tri-glial cocultures. Furthermore, the 21-day non-inducible neuron-tri-glial cocultures included neurons (NeuN, βIII-tubulin), astroglia (GFAP), microglia (Iba-1), oligodendrocyte progenitor cells ( PDGFRA , SOX), pre-myelinating oligodendrocytes (O1, O4, SOX10), and mature oligodendrocytes (MBP, SOX10), as well as an unknown vascular-related population. Meanwhile, mature oligodendrocytes form myelin sheaths that enclose neuronal axons. Moreover, to determine whether the oligodendrocyte lineage was vulnerable to nutrient and immune challenges, our data revealed that serum supplementation promoted oligodendrocyte lineage progression. In response to lipopolysaccharide treatment, the inflammatory factors TNF-α and IL-10 were produced in a time- and dose-dependent manner, along with microglia-dependent impairment of oligodendrocyte maturation, indicating that microglia are immunocompetent and involved. This study presents an innovative primary, high-throughput, non-inducible neuron-tri-glial coculture system that incorporates neurons, astroglia, microglia, and oligodendrocyte lineage cells. This system more closely mimics the in vivo brain environment and is suitable for mechanistic investigations and drug screening focused on myelin plasticity and oligodendrocyte lineage progression.