Nestin Reporter Transgene Labels Multiple Central Nervous System Precursor Cells

Embryonic neuroepithelia and adult subventricular zone (SVZ) stem and progenitor cells express nestin. We characterized a transgenic line that expresses enhanced green fluorescent protein (eGFP) specified to neural tissue by the second intronic enhancer of the nestin promoter that had several novel features. During embryogenesis, the dorsal telencephalon contained many and the ventral telencephalon few eGFP+ cells. eGFP+ cells were found in postnatal and adult neurogenic regions. eGFP+ cells in the SVZ expressed multiple phenotype markers, glial fibrillary acidic protein, Dlx, and neuroblast-specific molecules suggesting the transgene is expressed through the lineage. eGFP+ cell numbers increased in the SVZ after cortical injury, suggesting this line will be useful in probing postinjury neurogenesis. In non-neurogenic regions, eGFP was strongly expressed in oligodendrocyte progenitors, but not in astrocytes, even when they were reactive. This eGFP+ mouse will facilitate studies of proliferative neuroepithelia and adult neurogenesis, as well as of parenchymal oligodendrocytes

Characterization of Oligodendroglial Populations in Mouse Demyelinating Disease Using Flow Cytometry: Clues for MS Pathogenesis

<div><p>Characterizing and enumerating cells of the oligodendrocyte lineage (OLCs) is crucial for understanding demyelination and therapeutic benefit in models of demyelinating disease in the central nervous system. Here we describe a novel method for the rapid, unbiased analysis of mouse OLCs using flow cytometry. The assay was optimized to maximize viable yield of OLCs and maintain OLC antigen integrity. Panels of antibodies were assembled for simultaneous analysis of seven antigens on individual cells allowing for characterization of oligodendroglial cells throughout the lineage. We verified the utility of the assay with cultured OLCs and through a time course of developmental myelination. Next we employed the assay to characterize OLC populations in two well-characterized models of demyelination: cuprizone-induced demyelination and experimental autoimmune encephalomyelitis (EAE). In EAE we observed a dramatic loss of mature oligodendrocytes coincident with a dramatic expansion of oligodendrocyte progenitors cells (OPCs) at the onset of disease suggesting an attempt of the host to repair myelin. This expanded OPC pool was maintained through remission and relapse suggesting an arrest in differentiation in the face of the chronic autoimmune T cell-mediated inflammatory response. These robust, reproducible changes in OLCs through disease provide a rapid quantitative global analysis of myelin-producing cells in the adult mouse brain and important information regarding effects of disease on oligodendroglial proliferation/differentiation which is useful for defining the pathogenesis and therapy of MS.</p></div

Flow cytometry for oligodendroglial analysis.

<p>Flow cytometry allows for rapid analysis of eight or more proteins expressed on individual cells. Cells are streamed through a narrow flow cell sequentially and interrogated individually, thus largely static organs such as the brain must be dissociated into individual cells prior to analysis. Cells are stained with a cocktail of antibodies, each conjugated to a unique fluorochrome. Because of distinct proteins changes that occur during the maturation of the oligodendrocyte, antibodies can be utilized to characterize cells throughout the oligodendrocyte lineage. Thousands of cells per second are streamed through the flow cytometer, excited by laser light, and emitted fluorescence is directed through filters to photomultiplier tubes specific for each fluorochrome. For subsequent analysis, single cells are distinguished from small debris using forward (FSC) and side (SSC) scatter of light. Live cells are distinguished from dead cells using a live/dead dye, and CNS resident cells are distinguished from residual hematopoietic cells using a CD45 antibody. The entire oligodendrocyte lineage, from early progenitor to mature oligodendrocyte, can be analyzed and quantified.</p

Characterizing oligodendroglial population changes through cuprizone-induced demyelination.

<p>C57BL/6 mice were fed 0.2% cuprizone chow for five weeks and returned to a normal diet for a sixth week. A cohort of mice was sacrificed weekly throughout the disease course, brains were extracted, and CNS processed for flow cytometry (n = 5). Naïve mice were concurrently assayed at each time point for standardization (n = 3). (<b>A</b>) Cells were first gated by forward and side scatter then single cells were gated. Live cells were next gated, and hematopoietic cells excluded by gating on CD45^-/low cells. (<b>B</b>) From the CNS resident cells, oligodendroglial populations were defined: A2B5⁺PDGFRα⁺ early OPCs, PDGFRα⁺O4⁺ intermediate OPCs, O4⁺O1⁺ pre-myelinating oligodendrocytes, and O1⁺GALC⁺ mature oligodendrocytes. Mature oligodendrocytes (<b>C</b>), pre-myelinating oligodendrocytes (<b>D</b>), early OPCs (<b>E</b>), and late OPCs (<b>F</b>) were compared to naïve mice at each time point. CD45^high peripheral immune cells (<b>G</b>) and CD45^low microglia (<b>H</b>) were also quantified. Error bars indicate SD of mice per time point. *<i>P</i>≤0.05; **<i>P</i>≤0.01; ***<i>P</i>≤0.001 for post-hoc analysis. Data are representative of two independent experiments.</p

Oligodendroglial lineage analysis during development.

<p>Postnatal day four through ten C57BL/6 mice were sacrificed, brains extracted, and CNS tissue prepared for flow cytometry (n≥3/time point). Cells were stained with an antibody panel consisting of anti-A2B5, -NG2, -GALC, and -CD45 and analyzed by flow cytometry to characterize changes in oligodendroglial populations. (<b>A</b>) To examine CNS resident cells, single cells were gated, followed by live cells, followed by CD45⁻ gate. The GALC⁺ mature oligodendrocyte population was defined, and the A2B5⁺ and NG2⁺ oligodendrocyte progenitor cell (OPC) populations were defined from the GALC⁻ gate. (<b>b</b>) Representative oligodendroglial analysis from postnatal day 7 and 10 mice. Upper histograms illustrate GALC⁺ mature oligodendrocyte staining. Lower plots indicate A2B5⁺NG2⁻ early OPC and A2B5⁺NG2⁺ intermediate OPC populations. (<b>C</b>) Total cell yields from brains of individual mice, day 4 vs. day 10, <i>P</i> = 0.0013. (<b>D</b>) A2B5⁺NG2⁻GALC⁻ early OPCs (circles), A2B5⁺NG2⁺GALC⁻ intermediate OPCs (squares), and A2B5⁻NG2⁻GALC⁺ mature oligodendrocytes (triangles) are depicted. Error bars represent SD of mice per time point. Early OPCs: day 4 vs. 7, <i>P</i>≤0.001; day 7 vs. 10, <i>P</i>≤0.01. Intermediate OPCs: day 7 vs. 8, <i>P</i>≤0.001; day 8 vs. 10, <i>P</i>≤0.001. Mature oligodendrocytes: day 8 vs. 10, <i>P</i>≤0.05. Data are representative of three independent experiments.</p

Characterizing oligodendroglial populations through relapsing-remitting EAE.

<p>SJL/J mice were immunized with PLP_139–151 and scored daily for clinical disease. A cohort of mice was sacrificed at disease onset, peak, remission, and relapse, and spinal cords were analyzed by flow cytometry (n = 5). Naïve mice were also assayed at each time point for standardization across days (n = 3). (<b>A</b>) Cells were distinguished from debris by forward and side scatter then singlet cells were gated. Live cells were gated by dead cell exclusion, and CNS resident cells were identified as CD45⁻ or CD45^low. (<b>B</b>) Oligodendroglial cells were defined by double positive staining: A2B5⁺PDGFRα⁺ early OPCs, A2B5⁺NG2⁺ intermediate OPCs, NG2⁺O4⁺ late OPCs, O4⁺MOG⁺ pre-myelinating oligodendrocytes, and GALC⁺MOG⁺ mature oligodendrocytes. (<b>C</b>) The complete oligodendroglial lineage is illustrated for the naïve spinal cord. Oligodendroglial populations at each stage of disease were compared to naïve spinal cord. The table indicates number and percentage of CD45^-/low CNS resident cells ± SD per group of mice. (<b>D</b>) Clinical relapsing-remitting disease course and sacrifice points. (<b>E,F</b>) CD45^high infiltrating immune cells and CD45^low microglia in the spinal cord were also quantified throughout EAE disease. *<i>P</i>≤0.05; **<i>P</i>≤0.01; ***<i>P</i>≤0.001 for post-hoc analysis. Data are representative of three independent experiments.</p