Characterization of a murine mixed neuron-glia model and cellular responses to regulatory T cell-derived factors

Abstract

One of the unmet clinical needs in demyelinating diseases such as Multiple Sclerosis (MS) is to provide therapies that actively enhance the process of myelin regeneration (remyelination) in the central nervous system (CNS). Oligodendrocytes, the myelinating cells of the CNS, play a central role in remyelination and originate from oligodendrocyte progenitor cells (OPCs). We recently showed that depletion of regulatory T cells (Treg) impairs remyelination in vivo, and that Treg-secreted factors directly enhance oligodendrocyte differentiation. Here we aim to further characterize the dynamics of Treg-enhanced oligodendrocyte differentiation as well as elucidate the cellular components of a murine mixed neuron-glia model.Murine mixed neuron-glia cultures were generated from P2–7 C57BL/6 mice and characterized for percentage of neuronal and glial cell populations prior to treatment at 7 days in vitro (div) as well as after treatment with Treg-conditioned media at multiple timepoints up to 12 div.Mixed neuron-glia cultures consisted of approximately 30% oligodendroglial lineage cells, 20% neurons and 10% microglia. Furthermore, a full layer of astrocytes, that could not be quantified, was present. Treatment with Treg-conditioned media enhanced the proportion of MBP+ oligodendrocytes and decreased the proportion of PDGFRα+ OPCs, but did not affect OPC proliferation or survival. Treg-enhanced oligodendrocyte differentiation was not caused by Treg polarizing factors, was dependent on the number of activation cycles Treg underwent and was robustly achieved by using 5% conditioned media.These studies provide in-depth characterization of a murine mixed neuron-glia model as well as further insights into the dynamics of Treg-enhanced oligodendrocyte differentiation.Electronic supplementary materialThe online version of this article (10.1186/s13041-018-0367-6) contains supplementary material, which is available to authorized users.

Dynamic CCN3 expression in the murine CNS does not confer essential roles in myelination or remyelination

“…S4 A and B). This suggests the previously reported (9) positive effect of CCN3 on OPC differentiation in mixed glial cultures may have been indirect and potentially involved other CNS cell types such as astrocytes, neurons, microglia, pericytes, or even small populations of endothelial cells found within this model (45). Our findings highlight the complexity of CCN biology.…”

“…Pure OPC (POPC) cultures were generated from male and female WT P5-9 mice as previously described ( 45 ). At day 9 in culture, PDGFαα and NeuroTrophin 3 (NT3) were withdrawn to allow oligodendrocyte differentiation and cells were stimulated with 5% Treg-CM, 5% CCN3-depleted Treg-CM, 5% IgG isotype-depleted Treg-CM, or controls for up to 3 d. Purity was assessed in random cultures at 3–5 d after setup and was over 93%.…”

Oligodendrocytes in Development, Myelin Generation and Beyond

“…The methods described above (summarised in Table 1) entail the use of an isolated population. Other methods have been developed to mimic the complexity of the environment in which OPC and oligodendrocytes reside, such as mixed glia cultures [144], organotypic brain slices [145], and iPSC derived organoids [146]. These models are useful to study the impact of other cell types on OPC and oligodendrocytes, to decipher any indirect effect exerted by other CNS cells and to complement in vivo studies.…”

Multiple Sclerosis: Lipids, Lymphocytes, and Vitamin D

“…BHMT1 uses Zn 2+ ion as a cofactor and betaine as the methyl donor, The demonstration that 1,25(OH)2D3 rapidly increases Helios and promotes CD4 + Helios + FoxP3 + Treg cell dominance is significant in the context of emerging evidence that Treg cells, originally named for their immunoregulatory role, also have myelin regenerative functions [204][205][206]. Oligodendrocyte differentiation and remyelination were impaired in Treg-deficient mice and these defects were rescued by adoptive Treg cell transfer.…”