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Abstract
The molecular requirements for human myelination are incompletely defined, and further study is needed to fully understand the cellular mechanisms involved during development and in demyelinating diseases. We have established a human co-culture model to study myelination. Our earlier observations showed that addition of human γ-carboxylated growth-arrest-specific protein 6 (Gas6) to human oligodendrocyte progenitor cell (OPC) cultures enhanced their survival and maturation. Therefore, we explored the effect of Gas6 in co-cultures of enriched OPCs plated on axons of human fetal dorsal root ganglia explant. Gas6 significantly enhanced the number of myelin basic protein-positive (MBP+) oligodendrocytes with membranous processes parallel with and ensheathing axons relative to co-cultures maintained in defined medium only for 14 days. Gas6 did not increase the overall number of MBP+ oligodendrocytes/culture; however, it significantly increased the length of MBP+ oligodendrocyte processes in contact with and wrapping axons. Multiple oligodendrocytes were in contact with a single axon, and several processes from one oligodendrocyte made contact with one or multiple axons. Electron microscopy supported confocal Z-series microscopy demonstrating axonal ensheathment by MBP+ oligodendrocyte membranous processes in Gas6-treated co-cultures. Contacts between the axonal and oligodendrocyte membranes were evident and multiple wraps of oligodendrocyte membrane around the axon were visible supporting a model system in which to study events in human myelination and aspects of non-compact myelin formation.
Highlights
Myelination is essential for efficient ensheathment and insulation of axons allowing for enhanced axonal conductance and transport
While a great deal of our understanding of myelination comes from studying rodent model systems, there is an incomplete understanding of how efficient myelination is achieved during human development, and reparative processes occurring subsequent to neurologic diseases in the central nervous system (CNS)
Unless otherwise noted, enriched oligodendrocyte progenitor cell (OPC) purified from mixed glial cultures were immediately plated without further culturing on to the dorsal root ganglion (DRG) explant at 1×105 cells/well in defined medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) high-glucose medium (Gibco 11995) supplemented with 2 % B27 (Gibco 17504), 1 % N2 (Gibco 17502), 20 ng/ml recombinant human brainderived neurotrophic factor (BDNF) (Peprotech), 20 ng/ml
Summary
Myelination is essential for efficient ensheathment and insulation of axons allowing for enhanced axonal conductance and transport. While a great deal of our understanding of myelination comes from studying rodent model systems, there is an incomplete understanding of how efficient myelination is achieved during human development, and reparative processes occurring subsequent to neurologic diseases in the central nervous system (CNS). This lack of knowledge has hampered our understanding of how to optimize treatment for humans. Our goal was to combine growth factors that enhance oligodendrocyte progenitor cell (OPC) survival and maturation to generate a human co-culture model that expresses myelin-synthesizing rrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrr
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