A culture system to study oligodendrocyte myelination processes using engineered nanofibers

Abstract

Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive for myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment prior to differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination as well as provide valuable insight into the processes involved in remyelination.

Myelination and mTOR

“…Rather, CNS myelination seems to depend on an intrinsic myelination program of OLs, in combination with extrinsic signals from growth factors and from the electrical activity of axons. Differently from SCs, OLs retain the potential to produce myelin also when axons are fixed with paraformaldehyde or substituted with inert artificial fibers (Lee et al, 2012; Rosenberg, Kelland, Tokar, De la Torre, & Chan, 2008). These observations suggested a “default” myelination program, intrinsic to OLs, that does not depend on other axonal signals than a threshold of axonal caliber.…”

Developmental pruning of early-stage myelin segments during CNS myelination in vivo

“…Furthermore, segments wrapping larger axons will win and ones wrapping smaller axons will lose, raising the possibility that myelin pruning exhibits a competitive property. This may explain why large axons recruit glial wrapping early and receive more myelin layers than small axons [1,4,10]. During the pruning process, NRG-ErbB signaling may serve as a competitive signal at extending myelin segments or a pruning signal at pruning segments.…”

A Modular Assembly of Spinal Cord–Like Tissue Allows Targeted Tissue Repair in the Transected Spinal Cord

“…For example, although the singly cultured WMLT module exhibited a potential of forming myelin as evidenced by the detection of MBP immunoreactivity, there was a paucity of multilayered myelin sheath as observed under the electron microscopy, suggesting the structural immaturity of the oligodendrocytes. This might highlight the importance of axonal inputs in guiding the myelination process although the initiation of myelination could be independent from axon 25. Similarly, neurons in the singly cultured GMLT presented several immature features, as manifested by the synapse ultrastructure.…”

“…Nestin immunoreactivity was assessed and confirmed for all the neurospheres. Oligodendrocyte precursor cells (OPCs) were obtained according to the published protocols with slight modifications 25, 33. Briefly, NSCs at passage 2 were plated on polylysine‐coated culture dishes and cultured with DMEM/F12 containing 10 ng mL −1 platelet‐derived growth factor AA (PDGF‐AA, Life Technologies, USA), 10 ng mL −1 bFGF (Life Technologies, USA), 30 ng mL −1 triiodothyronine (T3, Sigma‐Aldrich), and 1% fetal bovine serum (FBS, Gibco) for 3 days.…”