Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Abstract

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines.Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification [1][2][3] . While many methods offer success, most require extensive culture times and/or costly equipment/ reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy & de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies.While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published [4][5][6] , a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

High-mobility group box-1 as an autocrine trophic factor in white matter stroke

“…Primary OL and microglial cell cultures were prepared from cortices of postnatal day 0 (P0) or P1 newborn wild-type and/or TLR2 (−/−) mice as previously described, with some modifications (49)(50)(51). The animal protocols were approved by Ajou University Institutional Animal Care and Use Committee.…”

Integrin-Linked Kinase Regulates Process Extension in Oligodendrocytes via Control of Actin Cytoskeletal Dynamics

“…OL cultures and OL/dorsal root ganglion neuron (DRGN) cocultures were derived as described previously (O'Meara et al, 2011b). For Rho kinase (ROCK) inhibition, compound Y-27632 (C 14 H 21 N 3 O) was added to each well at a final concentration of 10 M daily for 6 d. For control wells, an equal volume of water (vehicle) was added.…”

“…TAT-Cre (Excellgen) was added to mixed glial cultures at a concentration of ϳ2-5 M for 1-4 h. The following day, the efficiency of the recombination was qualitatively assessed by EGFP fluorescence. OPCs were extracted from the mixed glial cultures and seeded onto Ln-2 substrate 72 h after TATCre administration as described previously (O'Meara et al, 2011b).…”

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

“…For example, oligodendrocyte marker O4 may be used as an antigen to purify intermediate stage oligodendrocyte precursors using magnetic beads 20 . As an alternative to magnetic-based methods, OPCs can be enriched from 9 day old mixed glial cultures containing astrocytes and microglia by hi-speed orbital shaking and differential adhesion methods 21 . Furthermore, immuno-panning procedures which incorporate both negative and positive cell selection may be used if culture purity is preferred over high cell yields 22 .…”