Glial cells that express the NG2 proteoglycan and the ␣ receptor for PDGF (NG2 cells, polydendrocytes) make up the fifth major cell population that serves as oligodendrocyte progenitor cells in the postnatal CNS. Although recent studies have suggested differences in their proliferation and oligodendrocyte differentiation in gray and white matter, the mechanism underlying the observed differences has been unclear. Using organotypic slice cultures from the forebrain and cerebellum of early postnatal NG2creBAC:ZEG mice, we have compared basal and growth factor-induced proliferation of NG2 cells in gray and white matter. NG2 cells in white matter exhibited greater proliferative response to PDGF AA than those in gray matter. Heterotopic slice transplant and explant cultures suggested intrinsic mechanisms for the differential proliferative response of gray and white matter cells. Additionally, younger white matter NG2 cells showed a more robust proliferative response to PDGF. Basal and PDGF-induced proliferation of gray and white matter NG2 cells was largely dependent on Wnt/␤-catenin and phosphatidylinositol 3-kinase acting through the mammalian target of rapamycin pathway and not through ERK. These data uncover a previously unrecognized divergence between gray and white matter NG2 cells in the developing brain in their proliferative response to PDGF.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

Background: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses. Study Design and Methods: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization. Results: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG.

Oligodendrocyte precursor cells (OPCs), also known as NG2 cells or polydendrocytes, are distributed widely throughout the developing and mature central nervous system. They remain proliferative throughout life and are an important source of myelinating cells in normal and demyelinating brain as well as a source of glioma, the most common type of primary brain tumor with a poor prognosis. OPC proliferation is dependent on signaling mediated by platelet‐derived growth factor (PDGF) AA binding to its alpha receptor (PDGFRα). Here, we describe a group of structurally related compounds characterized by the presence of a basic guanidine group appended to an aromatic core that is effective in specifically repressing the transcription of Pdgfra but not the related beta receptor (Pdgfrb) in OPCs. These compounds specifically and dramatically reduced proliferation of OPCs but not that of astrocytes and did not affect signal transduction by PDGFRα. These findings suggest that the compounds could be further developed for potential use in combinatorial treatment strategies for neoplasms with dysregulated PDGFRα function.

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