GENESDEV/2007/ Supplementary Figure 1 Elkabetz et al.,

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12 SUPPLEMENTARY MATERIALS: SUPPLEMENTARY METHODS: Clonal density assays: Clonal assays were performed using GFP marked hesc derived R-NSCs co-cultured with unmarked stage-matched R-NSCs as feeder cells. This approach was required as single cells in the absence of R-NSC feeders did not survive compatible with the requirement for cell-to-cell contact typical for epithelial cells. RU1-eGFP-derived R-NSCs were sorted for Forse1 + /N- Cadherin high and seeded at clonal density on unsorted stage-matched R-NSCs derived from WA-09-derived. At 8 days after plating cultures were dissociated to single cell density followed by replating and expansion for an additional 8 21 days (P3) prior to assessing the formation of GFP+ rosettes. Efficiency of rosette formation in Forse1+ versus Forse1- cells was determined at a density of 1 RU1-eGFP cell/500 H9 [WA-09] cells. Antibodies used in this study: Monoclonal mouse antibodies: AQP4 (Abcam; 1:500), CD133 (Miltenyi Biotec; 1:50), En1 (DSHB; 1:75), Forse1 (DSHB; 1:75), GFAP (Chemicon; 1:500), HB9 (DSHB; 1:75), Human nuclear antigen (hna) (Chemicon; 1:100), Ki-67 (Dako, 1:100), MAP2 (Sigma; 1:200), Msx1 (DSHB; 1:75), N-cadherin (Sigma; 1:100), Nestin (Neuromics; 1:500), Nkx2.1 (Signet; 1:1), Oligodendrocyte marker 04 (Chemicon; 1:75), (Pax6 (DSHB; 1:75), PLZF (Calbiochem; 1:50), S100B (BD Biosciences, 1:1000), Tuj1 (Covance; 1:500), Vimentin-Cy3 (Sigma; 1:200), 3CB2 (DSHB; 1:75) and ZO-1 (BD Biosciences; 1:100). Monoclonal rat antibodies: anti-brdu (Abcam; 1:40). Polyclonal rabbit antibodies: BF-1 (kindly provided by E. Lai; 1:1000), βiii-tubulin (Covance; 1:1000), Dach1 (ProteinTech; 1:100), Olig1 (Chemicon; 1:200), Phospho Histone H3 (PH3) (Upstate; 1:1000), Sox1 (kindly provided by R. Lovell-Badge; 1:500), Sox2 (Abcam; 1:200), Zic1 (Novus Biological; 1:100) and ZO-1 (Zymed; 1:100). Polyclonal guinea-pig and goat antibodies: HoxA5 (kindly provided by T. Jessell), ChAT (Chemicon 1:200). Gobal gene expression analyses Venn Diagram Venn diagram for the three NSC samples Forse1+ P1 R-NSCs, Forse1- R-NSCs, and NSCs FGF/EGF was established using the R-statistical system and the Bioconductor microarray analysis packages. The raw Affymetrix cell files were normalized and quantitated using the GC- RMA package and the logarithm (base 2) of the expression levels were used in the subsequent analysis. To determine genes that were differentially expressed between each of the 3 differentiated samples versus undifferentiated hescs, a variant of the t-test was used as implemented in the LIMMA package from Bioconductor 1. This improves robustness of variance estimate in small sample sizes. To account for multiple testing, we used the False Discovery Rate method and applied a cutoff of 0.05 to each list. Transcripts expressed at least 5 times 1

13 higher in any of the differentiated samples compared to undifferentiated hescs were included in Venn diagram. Calculation of rosette score ( effective distance ) Maintenance of R-NSC marker profile under various treatment regimes rosette score was estimated by calculating the effective distance of each treatment condition on a vector between P1 R-NSC and NSC FGF/EGF state. To determine the relative distance and position (sign) along a linear axis for each treatment condition, we used the set of genes that were specifically expressed in P1 R-NSCs (298 genes; see Fig. 4b). Using the log (base 2) expression levels of the 298 genes, we computed the vector between Rosette and NSC state; x=nsc-rosette. This vector gives us a direction and distance in this gene expression space. We then took the vectors from P2 and P3 R-NSCs treated with SHH/FGF8, and P3 R-NSCs treated with SHH/Dll/Jag and projected them unto the NSCs FGF/EGF vector to determine the relative position of these states compared with the R-NSC state. Negative distance values indicate loss, positive values indicate and increase in rosette markers. To give a meaningful unit of distance along this vector we divided by the combined standard deviation of the NSC and Rosette replicate samples. In more precise terms we did the following. Let: x abc = (g 1,g 2,...,g 298 ) be the coordinates in the space of gene expression levels (log base 2) for the 298 genes specifically expressed in P1 R-NSCs. So xp1r NSC is the coordinates of the P1 R-NSC state and x FGF / EGF is the coordinates of the NSC FGF/EGF state and similarly for the other states. The NSC vector between two states is then defined as: v = x x FGF / EGF 1 FGF / EGF P1 R NSC, NSC P R NSC NSC Then the normalized distances ( rosette score ) are defined as: D typei v = SD v 1 P1 R NSC, R NSCregimen i FGF / EGF P1 R NSC, NSC 2 v FGF / EGF P1 R NSC, NSC 2 2 where SD = ( σp1r NSC+ σ FGF / EGF ) 2 (σ NSC X is the standard deviation of the X replicates) and the numerator is just the normal dot product between vectors and the denominator is the square of the vector length. 2

14 LEGENDS SUPPLEMENTARY IMAGES Supplementary Figure 1 Neuroepithelial and rosette features in hesc progeny using the SFEB induction protocol. Representative phase contrast images are presented. day2: Undifferentiated hescs grown as floating aggregates for 8 days and plated on Po/Lam-coated dishes for 2 days exhibit epithelial character similar to those of ES cells; day5: At 5 days after plating neuroepithelial feature emerge with cells exhibiting columnar cell shape. day 7: Cells show increased cell polarity and become radially arranged forming neural rosettes. day 40: Following FGF2/EGF mediated expansion and passaging in vitro, cells lose rosette structure and epithelial organization. Scale bar corresponds to 50 μm. Supplementary Figure 2 Forse1 marks an epitope on RA-treated NT2/D1 embryonal carcinoma cell line. The undifferentiated hesc marker SSEA-4 is presented on the surface of undifferentiated NT2/D1 cell line, but greatly reduced after RA-mediated neural differentiation (20 days of 10μM RA). In contrast, Forse1 marks a surface epitope expressed upon RAmediated neural induction of NT2/D1 cells but absent in the undifferentiated stage. Scale bar corresponds to 50 μm. Supplementary Figure 3 The induction of Forse1 expression during neural rosettes formation in hesc progeny. The Forse1 antigen is not presented on the surface of hescs at least until day 7 of neural induction (upper left panel). Forse1 expression subsequently increases gradually and by day 21 covers a large proportion of all hesc derived neural colonies (upper right and lower left panels). Scale bar corresponds to 100 μm in middle panel and 333 μm in left and right panels. Supplementary Figure 4 Forse1 co-localizes with neuroepithelial, radial glial and anterior CNS expression markers. Immunocytochemical analysis of P2 R-NSCs shows that cells expressing Forse1 also express neuroepithelial markers such as N-cadherin, radial glial markers such as vimentin and 3CB2, and anterior neural markers such as Otx2. Scale bar corresponds to 50 μm. Supplementary Figure 5 Isolation of R-NSCs by Forse1. P1 R-NSCs were dissociated and labeled for Forse1 and N-cadherin followed by FACS mediated isolation of Forse1+/Ncadherin high and Forse1-/N-cadherin high populations. (a) FACS plot for Forse1 expression. (b) FACS plot for co-expression of Forse1 and N-cadherin. (c) Forse1 expression represents approximately 30% of P1 R-NSCs. (d) Analysis of cell morphology and Forse1-FITC expression of Forse1+ and Forse1- cell populations immediately following sort. (e) Immunocytochemical analysis confirms that the vast majority of P1 R-NSCs comprised by Forse1+ and Forse1- sorted populations express the neural precursor markers nestin and musashi-1. Scale bar corresponds to 33 μm in lower right images and 100 μm in lower left images. Supplementary Figure 6 Efficiency of rosette re-formation from purified Forse1+ and Forse1- rosette stage cells. To compare the efficiency of rosette formation between Forse1 + 3

15 and Forse1 - cells, we plated Forse1 + /N-cad high and Forse1 - /N-cad high cells at a ratio of 1:500 onto stage-matched WA-09 rosette progeny sorted for the same markers (a). These data showed that Forse1 + cells efficiently form rosettes independent of the Forse1 status of the WA- 09 rosette feeder cells. Forse1 - cells were capable of rosette formation albeit at a reduced efficiency, particularly when plated on Forse1 + WA-09 rosettes (b,c). Statistical analysis: Mean ± S.E.M.; *** p < 0.001, ** p < 0.01 (comparison between Forse1 + and Forse1 - cells cocultured on Forse1+ or Forse1- cells; ANOVA) Supplementary Figure 7 Loss of Forse1 and BF1 expression in response to RA-induced caudalization. (a) Analysis of Forse1 expression and rosette morphology (phase contrast) under control conditions or upon exposure to RA. (b) Immunocytochemistry for BF1 and HoxB4 shows concomitant loss of BF1 expression and induction of HoxB4 upon RA treatment. Scale bar in (b) corresponds to 25 μm in (a) and 50 μm in (b). Supplementary Figure 8 Immunocytochemical analysis of neural crest markers in Forse1 - and NSC FGF2/EGF progeny four weeks after transplantation into the adult rat striatum (Inset shows matched in vivo immunohistochemistry for unsorted P1 rosette progeny). Scale bar corresponds to 100 μm for all panels. Supplementary Figure 9 PLZF expression in R-NSCs. Immunocytochemical analysis for PLZF in undifferentiated hescs, in P2 R-NSCs after FACS mediated separation of Forse1+ and Forse1- progeny at P1, and in NSC FGF/EGF. Scale bar corresponds to 100 μm. Supplementary Figure 10 qrt-pcr analysis of top R-NSC markers. Selected list of R-NSC specific markers from Fig. 4c was subjected to qrt-pcr analysis. These data confirmed specific enrichment in Forse1+ and Forse1- R-NSCs versus undifferentiated hescs NSCs FGF/EGF. Supplementary Figure 11 Immunohistochemical analysis of R-NSC and NSC FGF2/EGF progeny four weeks after transplantation into the adult rat striatum. Expression of R-NSC markers was largely restricted to grafts of R-NSCs and not observed in grafts derived from NSCs FGF/EGF. Right panels: PH3/Ki67 analysis revealed evidence for interkinetic nuclear migration in grafts derived from R-NSCs but not in those derived from NSCs FGF/EGF. Scale bar in corresponds to 25 μm for right and left panels and to 50 μm for middle panels. REFERENCES: 1. Smyth,G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article 3 (2004). 4

Supplementary figures (Zhang)

Supplementary figures (Zhang) Supplementary figure 1. Characterization of hesc-derived astroglia. (a) Treatment of astroglia with LIF and CNTF increases the proportion of GFAP+ cells, whereas the percentage