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Tissue-Specific Stem Cells
Version of Record online: 27 NOV 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 12, pages 2796–2809, December 2012
How to Cite
Moreno-EstellÉs, M., GonzÁlez-Gómez, P., Hortigüela, R., Díaz-Moreno, M., San Emeterio, J., AL, C., FariÑas, I. and Mira, H. (2012), Symmetric Expansion of Neural Stem Cells from the Adult Olfactory Bulb Is Driven by Astrocytes Via WNT7A. STEM CELLS, 30: 2796–2809. doi: 10.1002/stem.1243
Author contributions: M.M.-E.: conception and design, collection and/or assembly of data, and data analysis and interpretation; R.H., P.G.-G., M.D.-M., J.S.E., and A.L.C.: collection and/or assembly of data; I.F.: conception and design and financial support; H.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, financial support, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS September 17, 2012.
- Issue online: 27 NOV 2012
- Version of Record online: 27 NOV 2012
- Accepted manuscript online: 17 SEP 2012 03:51PM EST
- Manuscript Accepted: 14 AUG 2012
- Manuscript Received: 27 MAR 2012
- Ministerio de Sanidad y Consumo (Fondo de Investigación Sanitaria [FIS]-PI06/0754 and PI09/2254)
- Ministerio de Ciencia e Innovación (SAF program, CIBERNED, RETIC Tercel)
- Generalitat Valenciana (Programa Prometeo)
- PFIS fellowship from Ministerio de Sanidad, Política Social e Igualdad
- “Sara Borrell” Postdoctoral fellowship from Ministerio de Ciencia e Innovación (MICINN)-FIS
- predoctoral fellowship FPU from Ministerio de Educación
Additional Supporting Information may be found in the online version of this article.
|sc-12-0303_sm_SupplFigure1.tif||17348K||Supplementary Figure 1: In vivo distribution of SVZ-NSCs co-expressing GFAP and SOX2. Coronal section through the SVZ from 2-m old wild-type (CD-1) mice, stained for GFAP (green) and SOX2 (red), as seen by confocal microscopy. Nuclear label DAPI is shown in blue. The dashed line delineates the collapsed ventricular cavity. Sp, septum. St, striatum. Scale bar: 50 μm|
|sc-12-0303_sm_SupplFigure2.tif||36597K||Supplementary Figure 2: Identification of CldU+ and IdU+ cells in the olfactory bulb. (A) Coronal section inmunostained for CldU (green) and NeuN (red). CIdU+ cells in the oGCL colocalize with the nuclear mature neuronal marker NeuN. Nuclear label DAPI is shown in blue. Scale bar: 20 μm. (B) Coronal section inmunostained for IdU (red) and DCX (magenta). Most IdU+ cells located in the SEL (or very close to it) co-localize with the neuroblast marker DCX. Nuclear label DAPI is shown in blue. Scale bar: 50 μm|
|sc-12-0303_sm_SupplFigure3.tif||2081K||Supplementary Figure 3: The OB cells with spherogenic capacity are GLAST positive. (A) Percentage of GFAP+SOX2+ cells that are also GLAST+ in the SEL-iGCL of 2-m old wild-type (CD-1) mice (average ± sem, n = 4 animals), as determined by confocal microscopy. (B) After magnetic separation, positive and negative GLAST (ACSA-1) fractions derived from the OB tissue were stained with secondary Alexa 488 antibody and analyzed by flow cytometry. The result demonstrates that the GLAST+ fraction was highly enriched in cells expressing high levels of GLAST. (C) Summary table showing data of three independent experiments performed. The GLAST negative fraction did not form neurospheres under the experimental conditions that were used. The sphere formation efficiency of the GLAST positive fraction was 0.26 ± 0.05 % (average ± sem, n = 3 independent experiments).|
|sc-12-0303_sm_SupplFigure4.tif||8734K||Supplementary Figure 4: Characterisation of the glial cultures. Cultures from postnatal mice were highly enriched in Glial Fibrillary Acidic Protein (GFAP). Most cells displayed a protoplasmic morphology and were negative for the type C polyganglioside marker (A2B5), indicating they were type 1 astrocytes. Immunocytochemistry for GFAP is shown in red and for A2B5 is shown in green. Nuclear label DAPI is shown in blue. (A) Type 1 astrocyte, protoplasmic, GFAP positive and A2B5 negative. (B) Type 2 astrocyte, fibrous, GFAP positive and A2B5 positive. Scale bar: 20 μm|
|sc-12-0303_sm_SupplFigure5.tif||2556K||Supplementary Figure 5: Diffusible factors from adult OB and SVZ astrocytes regulate adult OB-NSC activity, while SVZ-NSCs are also regulated by astrocytic soluble factors. (A) Schematic drawing illustrating the indirect co-culture system. OB-NSCs were seeded in the upper transwell, while adult astrocytes were grown as feeder cells in the lower compartment. (B) Number of OB neurospheres obtained by plating equal numbers of cells dissociated from OB-NSC cultures in the presence of adult astrocytes from different brain areas or in the absence of astrocytes as a control (n = 3). (C) Average size of the OB neurospheres obtained in the adult glial co-culture (n = 3). (D) Schematic drawing illustrating the experimental setup for the conditioned medium assays. Adult SVZ-NSCs were treated with filtered serum-free medium from region-specific astrocytic cultures that had been grown for 3 days in vitro (DIV). The conditioned medium was supplemented with EGF and FGF-2. (E) Number of SVZ- neurospheres obtained by plating equal numbers of cells dissociated from SVZ-NSC cultures in conditioned medium from the astrocytes (n = 3). (F) Average size of the SVZ neurospheres generated in the conditioned medium (n = 3). Paired t-test: *p < 0.05, ** p < 0.01.|
|sc-12-0303_sm_SupplFigure6.tif||6102K||Supplementary Figure 6: OB-NSCs transcriptome expression analysis. Scanned images of the GEArray S Series Mouse Stem Cell Gene Arrays MM-601.2 membranes hybridised with biotinylated-dUTP cDNA probes prepared from RNA of OB-NSC cultures (n = 2). The results from the analysis are shown in Supplementary Table 5.|
|sc-12-0303_sm_SupplFigure7.tif||4562K||Supplementary Figure 7: OB-NSCs express Wnt signalling components. PCR validation for several Fzd genes, which code for Wnt receptors (A) and other essential components of both canonical and non-canonical WNT signalling (B) in three independent OB-neurosphere cultures.|
|sc-12-0303_sm_SupplFigure8.tif||1519K||Supplementary Figure 8: WNT7A increases the number of secondary SVZ-neurospheres. (A) Primary SVZ-neurosphere yield from 2-m old CD-1 mice untreated and treated with WNT7A 50ng/ml (n = 12). (B) Fold increase in the number of secondary SVZ-neurospheres grown in the presence of WNT7A 50 ng/ml (n = 7). (C) Self-renewal assay in which neurospheres formed in growth medium with or without WNT7A were dissociated to single cells and plated at low density in the absence of WNT7A. The WNT7A pre-treatment improved the self-renewal capacity of SVZNSCs. More secondary spheres were formed from the pre-treated cells (n = 2). Paired t-test: ** p < 0.01.|
|sc-12-0303_sm_SupplTable1.pdf||28K||Supplementary Table 1|
|sc-12-0303_sm_SupplTable2.pdf||40K||Supplementary Table 2|
|sc-12-0303_sm_SupplTable3.pdf||20K||Supplementary Table 3|
|sc-12-0303_sm_SupplTable4.pdf||34K||Supplementary Table 4|
|sc-12-0303_sm_SupplTable5.pdf||30K||Supplementary Table 5|
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