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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 24 MAR 2013
Copyright © 2012 AlphaMed Press
Volume 31, Issue 4, pages 652–665, April 2013
How to Cite
Srivastava, R., Kumar, M., Peineau, S., Csaba, Z., Mani, S., Gressens, P. and El Ghouzzi, V. (2013), Conditional Induction of Math1 Specifies Embryonic Stem Cells to Cerebellar Granule Neuron Lineage and Promotes Differentiation into Mature Granule Neurons. STEM CELLS, 31: 652–665. doi: 10.1002/stem.1295
Author contributions: R.S.: generation, characterization, differentiation of mouse stem cells, real-time PCR, immunocytochemistry, fluorescent microscopy, imaging, cell counting, collection, analysis and interpretation of data, and writing of material and methods section; M.K.: stem cells culture, differentiation, immunocytochemistry, immunohistochemistry, confocal microscopy, and imaging; S.P.: electrophysiological experiments and analyses; Z.C.: in vivo implantations and surgery; S.M.: conception of the study, data interpretation, and manuscript writing; P.G.: data interpretation, financial support, and final approval of manuscript; V.E.G.: conception and design of the study, Western blots, data analysis and interpretation, financial support, manuscript writing, and final approval of manuscript. S.M. and P.G. contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLS EXPRESS December 7, 2012.
- Issue online: 24 MAR 2013
- Version of Record online: 24 MAR 2013
- Manuscript Accepted: 23 OCT 2012
- Manuscript Received: 28 FEB 2012
- Institut National pour la Santé et la Recherche Médicale (INSERM, France)
- Centre National de la Recherche Scientifique (CNRS, France)
- Denis Diderot University (Paris7, France)
- National Brain Research Centre (Haryana, India)
- IFCPAR/CEFIPRA. Grant Number: project N°3803-3
- Department of Biotechnology. Grant Number: project N°BT/PR11653/MED/31/602008
- French National Research Agency. Grant Number: project ANR-09-GENO-007
- Princesse Grâce de Monaco Foundation
- Roger de Spoelberch Foundation
- Medical Research Council
Additional Supporting Information may be found in the online version of this article.
|sc-12-0198_sm_SupplFigure1.tif||424K||Supplemental Figure 1. Sustained expression of the pluripotency-related markers SSEA1 and Oct4 in control ESD3 cells and Math1-inducible clone 1.2. Scale bar = 20μm.|
|sc-12-0198_sm_SupplFigure2.tif||1592K||Supplemental Figure 2. (A): RT-PCR of markers of the three germ layers (endoderm (GATA4), mesoderm (Bmp2) and neurectoderm (Nestin, Pax6) in early EBs (before RA addition) and late EBs (RA-induced). Note that expression of both Nestin and Pax6 is strongly increased upon RA induction. (B): Real-time PCR analysis of En1 and NeuroD1 in late EBs and FD stage with (Dox) and without (Ctl) Math1 induction. Quantitative data are expressed as mean ± SEM for each group. (late EBs: n=11, En1 *** P<0.0001, NeuroD1 * P=0.0374. FD stage: n=11, En1 *** P=0.0013, NeuroD1 P=0.3403 (Mann–Whitney)). (C): Cell countings of Ki67 and cleaved caspase3 in late EBs and Neurospheres (NS) in Ctl and Dox cultures. Quantitative data are expressed as mean ± SEM for each group. (n=6, ** P=0.0022, (Mann–Whitney)).|
|sc-12-0198_sm_SupplFigure3.tif||1071K||Supplemental Figure 3. (A): Real-time PCR analysis of Tuj1, GFAP, MAP2 and Olig2 genes expression at FD stage in the presence of extrinsic factors (GF cocktail) with (Dox) and without (Ctl) Math1 induction. Quantitative data are expressed as mean ± SEM for each group. (B): Cell countings of En1, Zic2 and Pde1c at FD stage in the presence or absence of extrinsic factors (GF cocktail) in Ctl and Dox cultures. Quantitative data are expressed as mean ± SEM for each group. (n=6, En1 ** P=0.0022, ## P=0.022. Zic2 vs Tuj1 without factors * P=0.0260, Pde1c ** P=0.005 and ##P=0.022 (Mann– Whitney)).|
|sc-12-0198_sm_SupplFigure4.pdf||1024K||Supplemental Figure 4. Neural properties of clone 1.2 and comparison with ESD3 and ESB6 cell lines. (A): Cell countings of Tuj1, GFAP, MAP2 and Olig2 at FD stage in basal conditions for the three cell lines clone 1.2, ESD3 and ESB6. Quantitative data are expressed as mean ± SEM for each group (n=6). (B): Representative immunolabelings of Tuj1, MAP2, GFAP and Olig2 at FD stage in basal conditions for the three cell lines. Scale bar = 50μm|
|sc-12-0198_sm_SupplFigure5.pdf||1195K||Supplemental Figure 5. (A): Real time PCR and western blot analysis of Math1 expression upon Dox addition of clones 1.3 and 2.8 during (early+late) EBs stage (B): Cell countings of Tuj1, GFAP, MAP2 and Olig2 after immunocytochemistry at FD stage in Ctl and Dox cultures from clones 1.3 and 2.8. Like in clone 1.2, cells positive for Tuj1 and MAP2 are more numerous upon Dox addition and cells positive for GFAP are less. Quantitative data are expressed as mean ± SEM for each group (n=6, clone 1.3, ** P=0.0022, n=6, clone 2.8 ** P=0.0022 (Mann–Whitney)). (C): Representative immunolabelings at FD stage of Tuj1, MAP2, GFAP and Olig2 in Ctl and Dox cultures from clones 1.3 and 2.8. Scale bar = 50μm.|
|sc-12-0198_sm_SupplFigure6.pdf||1347K||Supplemental Figure 6. (A): Cell countings of cerebellar granule cell markers after immunocytochemistry at FD stage in the presence or absence of extrinsic factors (GF cocktail) in Ctl and Dox cultures from clones 1.3 and 2.8. Similar trends as described for clone 1.2 are observed. Quantitative data are expressed as mean ± SEM for each group. (clone 1.3, n=6, GABAa6r * P=0.0161, ** P=0.005, ## P=0.005, Zic1 * P=0.0129, ** P=0.0022, Pde1c ** P=0.005 in basal conditions, ** P=0.0022 in the presence of GF cocktail, ## P=0.005, MAP2 ## P=0.0022 (Mann–Whitney) and clone 2.8, n=6, GABAa6r and Zic1, ** P=0.0022, ## P=0.005, Pde1c * P=0.0161, ** P=0.0087, ## P=0.0022, MAP2 ## P=0.0022 (Mann–Whitney)). (B): Representative immuno-colabelings at FD stage of cerebellar granule cell markers in Dox-induced cultures with extrinsic factors (GF cocktail). As shown for clone 1.2, in clone 1.3 and clone 2.8 derived cultures a majority of Tuj1- or MAP2-expressing cells also express GABAa6r, Zic1 and Pde1c. (C): Mature neurons are able to generate spontaneous activity (upper graph, cl 1.3) and firing following 30mV depolarization (middle and lower graphs, cl 1.3 and 2.8 respectively).|
|sc-12-0198_sm_SupplFigure7.tif||919K||Supplemental Figure 7. Specificity of the immunostainings was checked using non-relevant primary antibodies that do not react with any antigen (mouse IgG1 isotype control (Abcam, ab126026) and rabbit IgG isotype control (Abcam, ab27478).|
|sc-12-0198_sm_SupplTable1.pdf||86K||Supplemental Table 1|
|sc-12-0198_sm_SupplTable2.pdf||78K||Supplemental Table 2|
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