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sc-12-0198_sm_SupplFigure1.tif424KSupplemental 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.tif1592KSupplemental 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.tif1071KSupplemental 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.pdf1024KSupplemental 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.pdf1195KSupplemental 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.pdf1347KSupplemental 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.tif919KSupplemental 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.pdf86KSupplemental Table 1
sc-12-0198_sm_SupplTable2.pdf78KSupplemental Table 2

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