Myelination and node of Ranvier formation on sensory neurons in a defined in vitro system

Abstract

One of the most important developmental modifications of the nervous system is Schwann cell myelination of axons. Schwann cells ensheath axons to create myelin segments to provide protection to the axon as well as increase the conduction of action potentials. In vitro neuronal systems provide a unique modality to study a variety of factors influencing myelination as well as diseases associated with myelin sheath degradation. This work details the development of a patterned in vitro myelinating dorsal root ganglion culture. This defined system utilized a serum-free medium in combination with a patterned substrate, utilizing the cytophobic and cytophilic molecules (poly)ethylene glycol (PEG) and N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA), respectively. Directional outgrowth of the neurites and subsequent myelination was controlled by surface modifications, and conformity to the pattern was measured over the duration of the experiments. The myelinated segments and nodal proteins were visualized and quantified using confocal microscopy. This tissue-engineered system provides a highly controlled, reproducible model for studying Schwann cell interactions with sensory neurons, as well as the myelination process, and its effect on neuronal plasticity and peripheral nerve regeneration. It is also compatible for use in bio-hybrid constructs to reproduce the stretch reflex arc on a chip because the media combination used is the same we have used previously for motoneurons, muscle and for neuromuscular junction (NMJ) formation. This work could have application for the study of demyelinating diseases such as diabetes induced peripheral neuropathy and could rapidly translate to a role in the discovery of drugs promoting enhanced peripheral nervous system (PNS) remyelination.

Development and validation of an in vitro model system to study peripheral sensory neuron development and injury

“…The use of differentiated peripheral sensory neurons for studying PNS development and injury depends on the ability of the in vitro cells to faithfully recapitulate the functional properties of in vivo sensory neurons particularly in terms of their electrophysiological properties and potential for myelination 14 , 25 , 26 . We first addressed the electrophysiological modality by whole-cell patch-clamp recordings at 35 DIV (Fig.…”

“…Since we observed this close association between the donor Schwann cells and the in vitro derived peripheral sensory neurons we next investigated whether this interaction triggered and supported the process of myelination. Myelin binding protein (MBP) is a major component of compact myelin sheaths 26 and we therefore assessed for the presence of this integral protein in combination with NEFH to demarcate any myelinating Schwann cells that ensheathed the peripheral sensory neurons (Fig. 6 ).…”

A phenotypic culture system for the molecular analysis of CNS myelination in the spinal cord

“…DETA also promotes long-term cell survival because it is non-digestible by matrix proteases secreted by cells [24, 33]. We have observed that DETA is a permissive substrate for a large number of cell types including neurons and glia and have already observed the myelination of sensory neurons [26] and motoneurons by Schwann cells on this substrate [23]. Hence, DETA lends itself to the long-term co-culture of the cell types required for efficient myelin formation.…”

Fetal bovine serum (FBS): Past – present – future

“…There are now also some examples that demonstrate that adaptation to FBS-free cell culture does not have a major impact on the robustness of cell culture systems or on the scientific outcome of the studies (Jukić et al, 2016;Oleaga et al, 2016;Rumsey et al, 2013;Stancescu et al, 2015). However, chemically-defined serum-free media have not yet been developed for every cell line or primary culture at this time (Bjare, 1992;Even et al, 2006;Gstraunthaler, 2003;Brunner et al, 2010).…”