Journal: Stem Cells and Development

Article Title: Purification, Visualization, and Molecular Signature of Neural Stem Cells

doi: 10.1089/scd.2015.0190

Figure Lengend Snippet: In vitro characterization of C1qR1 + cells. (A) Cells obtained from dissociated neurospheres were incubated with anti-Lewis-X (LeX), anti-Eng, anti-cKit, anti-Prom1, and anti-C1qR1 antibodies. Positive and negative cells for each marker were fluorescence-activated cell sorting (FACS)-sorted at 667 cells/mL into the culture medium containing epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). The number of neurospheres formed was counted after 5 days. “Unsorted cells” refers to sorting live cells with no gating on any marker. Data are presented as mean ± SEM from three experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. (B , C) Bright-field images of neurospheres generated from C1qR1 + and C1qR1 − cells. Scale bar = 100 μm. (D–F) Cell cycle analysis of neurosphere-dissociated cells stained with anti-C1qR1-phycoerythrin (PE); 4′-6-diamidino-2-phenylindole (DAPI) was used to separate the cells according to their cell cycle status (G 0 /G 1 phases from sorted [S]-G 2 /M phases). Analysis gates were set as illustrated in (D) . The C1qR1 + and C1qR1 − cells are colored blue and red , respectively. Ungated cells are colored black . The cell cycle profile of each population is shown in (E , F) . (G) The diameter of neurospheres generated from C1qR1 + and C1qR1 − cells was measured and divided into three categories, <50 μm, 50–100 μm, and >100 μm. The bar chart shows the percentage of neurospheres in each size category. (H) Unsorted, C1qR1 + , and C1qR1 − cells were plated at 667 cells/mL and neurosphere formation followed for 5 days. Neurospheres were then collected and triturated into single cells and reseeded at 667 cells/mL. The number of secondary neurospheres formed was counted 5 days later. (I) Single neurospheres derived from C1qR1 + and C1qR1 − cells grown under clonal conditions were differentiated and scored as tripotent, bipotent, or unipotent, according to the cell-type staining observed in each differentiated colony (see the section). Tripotent neurospheres contain cells that stained positive with glial fibrillary acidic protein (GFAP), ßIII-tubulin, and O4. Bipotent neurospheres contain cells that stained positive with GFAP/ßIII-tubulin or GFAP/O4. Unipotent neurospheres contain cells stained positive with GFAP alone. (J–M) Representative images of differentiated neurospheres stained for the lineage markers, O4 (oligodendrocyte; green ), βIII-tubulin (neuron; red ), and GFAP (astrocyte; gray ). Scale bar = 50 μm.

Article Snippet: Preincubation with a 10-fold excess of specific blocking peptide (1st BASE) against C1qR1 antibody was used as a negative control for this reaction.

Techniques: In Vitro, Incubation, Marker, Fluorescence, FACS, Generated, Cell Cycle Assay, Staining, Derivative Assay

Journal: Stem Cells and Development

Article Title: Purification, Visualization, and Molecular Signature of Neural Stem Cells

doi: 10.1089/scd.2015.0190

Figure Lengend Snippet: Immunohistochemical analysis of C1qR1 + cells. (A–H) Expression and localization of C1qR1 protein in the developing cerebral cortex at E14.5. Immunohistochemistry of cerebral cortex at low magnification (10×) (A–D) and high magnification (40×) (E–H) . Labeling for DAPI in (A) and (E) , Nestin in (B) and (F) , C1qR1 in (C) and (G) , and Merged in (D) and (H) . The boxes in (B–D) span the ventricular zone (VZ) from the apical to basal region and are shown at higher magnification in (F–H) . The inserts in (F–H) show optical zoom of the boxed areas . C1qR1 is localized in the membrane throughout the neural progenitor cells of the cortical ventricular zones (VZ and subventricular zone) with an intense staining in the VZ. The insert in (H) shows the cellular colocalization of C1qR1 and nestin (as seen in yellow ). Scale bars (A) = 100 μm and (E) = 40 μm. (I) Cells obtained from freshly dissociated embryonic brain were stained with anti-C1qR1-PE. Unsorted, C1qR1 + , and C1qR1 − cells were sorted and plated at 667 cells/mL. The number of primary neurospheres formed in the presence of EGF and FGF2 was counted 5 days later. Data are presented as mean ± SEM from three experiments. * P < 0.05, ** P < 0.01. (J) Bright-field image of secondary neurosphere generated from C1qR1 + cells. Scale bar = 100 μm, at 4× magnification. (K–N) C1qR1 + population gives rise to neurospheres that differentiate into oligodendrocytes (O4; green ), neurons (βIII-tubulin; red ), and astrocytes (GFAP; gray ). Scale bar = 50 μm.

Article Snippet: Preincubation with a 10-fold excess of specific blocking peptide (1st BASE) against C1qR1 antibody was used as a negative control for this reaction.

Techniques: Immunohistochemical staining, Expressing, Immunohistochemistry, Labeling, Membrane, Staining, Generated

Journal: Stem Cells and Development

Article Title: Purification, Visualization, and Molecular Signature of Neural Stem Cells

doi: 10.1089/scd.2015.0190

Figure Lengend Snippet: Characteristics of LeX/C1qR1-sorted cells. Distribution and sorting gates (R1–R4) of the neurosphere-dissociated cells in combined assays. (A) Negative control for LeX/C1qR1, (B) LeX/C1qR1, (C) negative control for Prom1/C1qR1, (D) Prom1/C1qR1. Four regions (R1: doubly positive; R2 and R3: singly positive; and R4: doubly negative) as well as the unsorted (all live cells) from each combined assay were sorted. (E , F) Bar chart showing the percentage of neurospheres generated per single sorted cell for each subpopulation after 14 days of culture. Data are presented as mean ± SEM from four experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. (G) Relative expression of neural stem cell (NSC)-specific genes in each of the FACS-sorted subpopulations from (A) was analyzed by quantitative RT-PCR. The fold change in the gene expression profile was compared with unsorted cells ( black bar ). RT-PCR, reverse transcription polymerase chain reaction.

Article Snippet: Preincubation with a 10-fold excess of specific blocking peptide (1st BASE) against C1qR1 antibody was used as a negative control for this reaction.

Techniques: Negative Control, Generated, Expressing, Quantitative RT-PCR, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction

Journal: Stem Cells and Development

Article Title: Purification, Visualization, and Molecular Signature of Neural Stem Cells

doi: 10.1089/scd.2015.0190

Figure Lengend Snippet: NSC frequency of LeX/C1qR1-sorted cells. (A–D) Embryo forebrain (E14.5) coronal sections were counterstained with DAPI ( blue ) (A) , nestin ( green ) (B) , and NogoR1 ( red ) (C) . Merged is shown in (D) . The boxes show the location of the inserts in (B–D) . The inserts show optical zoom of the boxed areas . The signals of the insets have been adjusted to visualize colocalization of LeX and C1qR1. Scale bar = 40 μm. (E ) Individual clonal neurospheres were differentiated on coverslips coated with PLL/laminin and scored as follows: tripotent (astrocytes, neurons, and oligodendrocytes), bipotent (astrocytes/neurons or astrocytes/oligodendrocytes), and unipotent (astrocytes only). Data are presented as mean ± SEM from three experiments. * P < 0.05, *** P < 0.001. Antibodies and methods as described in the section. (F) NSC frequency was calculated by multiplying the absolute neurosphere-forming units by the percentage of multipotent neurospheres.

Article Snippet: Preincubation with a 10-fold excess of specific blocking peptide (1st BASE) against C1qR1 antibody was used as a negative control for this reaction.

Techniques:

Journal: Stem Cells and Development

Article Title: Purification, Visualization, and Molecular Signature of Neural Stem Cells

doi: 10.1089/scd.2015.0190

Figure Lengend Snippet: Time-lapse imaging of neurosphere formation. LeX + /C1qR1 + cells were seeded at clonal density in a 96-well plate and imaged from the single-cell stage for 5 days. (A) Cells forming neurospheres. (B) Cells dying without division. (C) Cells dying after one division and (D) cells dying after two divisions. (E) Quantification of data presented in (A–D) . Scale bar = 10 μm (Mean ± SEM; n = 5). (F , G) LeX + /C1qR1 + cells were seeded at clonal density and left for 24 h. Cells were stained with LeX (or C1qR1), doublets imaged for LeX distribution, and scored for neurosphere formation after 5 days.

Article Snippet: Preincubation with a 10-fold excess of specific blocking peptide (1st BASE) against C1qR1 antibody was used as a negative control for this reaction.

Techniques: Imaging, Staining

Journal: Stem Cells and Development

Article Title: Purification, Visualization, and Molecular Signature of Neural Stem Cells

doi: 10.1089/scd.2015.0190

Figure Lengend Snippet: Distribution of LeX and C1qR1 in neurospheres. (A) Day 5 neurospheres derived from LeX + /C1qR1 + cells and grown under clonal conditions were labeled with LeX ( green ) or C1qR1 ( red ) antibodies and imaged on an laser scanning confocal microscope (LSCM) to reveal distribution patterns. Slices from one three-dimensional stack are shown. Quantification of the fluorescence intensity of LeX in two different neurospheres revealed a difference in fluorescence intensity between bright cells relative to other cells within the same neurosphere. (B) Differentiated neurospheres were labeled with lineage-specific markers, O4, βIII tubulin, and GFAP, and the potency of individual neurospheres was scored (Mean ± SEM; 60 neurospheres, n = 4; * P < 0.01).

Article Snippet: Preincubation with a 10-fold excess of specific blocking peptide (1st BASE) against C1qR1 antibody was used as a negative control for this reaction.

Techniques: Derivative Assay, Labeling, Microscopy, Fluorescence

Journal: Stem Cells and Development

Article Title: Purification, Visualization, and Molecular Signature of Neural Stem Cells

doi: 10.1089/scd.2015.0190

Figure Lengend Snippet: Single-cell messenger RNA (mRNA) profiling of LeX + /C1qR1 + and LeX + /C1qR1 − cells. (A) Forty-eight-dimensional mRNA profiling data were compressed to two dimensions by nonmetric multidimensional scaling (nMDS). Axes for the two dimensions are labeled as nMDS_1 and nMDS_2. Model-based clustering (Mclust) was then used to cluster cells based on the mRNA profile of 48 genes. Three clusters were derived—I, II, and III. Each point represents one cell. (B–D) Bar plots showing log 2-fold change in gene expression for genes that show significant change in expression ( P < 0.05) in a cluster in comparison with the remaining clusters combined. (E) The ratio of LeX + /C1qR1 + cells to LeX + /C1qR1 − cells and the percentage of LeX + /C1qR1 + cells and LeX + /C1qR1 − cells in each cluster were calculated. The genes that show significant changes in expression in each cluster are shown. Passage 2 cells were used for single-cell mRNA profiling.

Article Snippet: Preincubation with a 10-fold excess of specific blocking peptide (1st BASE) against C1qR1 antibody was used as a negative control for this reaction.

Techniques: Labeling, Derivative Assay, Gene Expression, Expressing, Comparison