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Additional Supporting Information may be found in the online version of this article.

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SC_12-0152_sm_supplFigure1.pdf659KSupplementary Figure 1 LDN/SB induces PAX6+ efficiently across pluripotent cell lines and this effect is due to directed differentiation rather than cell selection by cell death. A) Induction of Detroit and BJ-1 pluripotent cells with LDN/SB results in comparable percentage of PAX6+ cells to that of hES (Shef-1) B) Induction of PAX6 occurs evenly across the well of 96-well plate: composite of seven images. Sample BJ-1 cell line results showed C) Increase in the total number of cells (DAPI) during the time course of induction D) %of EthD-1+ cells in relation to the total cell number identified by DAPI staining across time points of induction.
SC_12-0152_sm_supplFigure2.tif560KSupplementary Figure 2 Chemical structure of SB431542 and tested SB analogues. 1- Cpd1-SB431542; 2- Cpd2; 3- Cpd3; 4- Cpd4; 5- Cpd5; 6- Cpd6; 7- Cpd7; 8- Cpd8; 9- Cpd9.
SC_12-0152_sm_supplFigure3.tif2347KSupplementary Figure 3 SB431542, LDN192189 and Dorsomorphin alone do not induce PAX6 expression. Concentration response curve shown in logarithmic scale and normalised to the response of positive control (LDN/SB) for both PAX6 and OCT4.
SC_12-0152_sm_supplFigure4A.pdf2989KSupplementary Figure 4 Analysis of Cpd2 kinase screen against 138 human protein kinases. A) 2 graphs showing % activity remaining for tested kinases at two concentrations 1μM and 100nM B) IC50 values (shown in summary table) were determined using 4 point dose response curve and are shown as average of technical duplicate.
SC_12-0152_sm_supplFigure4B.tif1611KSupplementary Figure 4 Analysis of Cpd2 kinase screen against 138 human protein kinases. A) 2 graphs showing % activity remaining for tested kinases at two concentrations 1μM and 100nM B) IC50 values (shown in summary table) were determined using 4 point dose response curve and are shown as average of technical duplicate.
SC_12-0152_sm_supplFigure5.pdf729KSupplementary Figure 5 Microarray analysis of a population of cells during the time course of induction. A) Plot of differentiation as defined by PC2 against time for induced and non-induced samples. The dashed lines represent sigmoidal lines of best fit B) Hierarchical clustering of samples based on the correlation of their global gene expression profiles. Colors in the heatmap indicate the degree of correlation between a pair of samples. The dendrograms are constructed by using 1 – R as the Euclidean distance between samples followed by single linkage clustering. The pink-red bar indicates the time of sample collection from early (white/pink) to late (burgundy). C) Plot of the first two components of a principal component analysis on the sample expression profiles. Clear clustering by time and by sample treatment can be seen. Differentiation towards ANE seems to correlate most closely with PC2. D) Box plot showing the distribution of the variances of the top most significantly differentially expressed genes (ANOVA P < 1e-24) across each sample group. The non-induced D2-8 group (U-D2-8) shows a significantly higher median variance amongst these genes when compared to all other groups. E) Variance of POU5F1 and PAX6 in each of the sample groups. Significantly higher variance can be seen in the noninduced D2-8 group. F) Enrichment of forebrain and hindbrain development specific gene sets amongst the most significantly differentially expressed (DE) genes. Hypergeometric tests were run to test for enrichment (dashed lines) in either gene set amongst DE genes chosen from an ANOVA using the P value cut off indicated on the x-axis. Dotted lines indicate the percentage of each gene set amongst the DE genes at each cut off.
SC_12-0152_sm_supplFigure5C-F.tif1981KSupplementary Figure 5 Microarray analysis of a population of cells during the time course of induction. A) Plot of differentiation as defined by PC2 against time for induced and non-induced samples. The dashed lines represent sigmoidal lines of best fit B) Hierarchical clustering of samples based on the correlation of their global gene expression profiles. Colors in the heatmap indicate the degree of correlation between a pair of samples. The dendrograms are constructed by using 1 – R as the Euclidean distance between samples followed by single linkage clustering. The pink-red bar indicates the time of sample collection from early (white/pink) to late (burgundy). C) Plot of the first two components of a principal component analysis on the sample expression profiles. Clear clustering by time and by sample treatment can be seen. Differentiation towards ANE seems to correlate most closely with PC2. D) Box plot showing the distribution of the variances of the top most significantly differentially expressed genes (ANOVA P < 1e-24) across each sample group. The non-induced D2-8 group (U-D2-8) shows a significantly higher median variance amongst these genes when compared to all other groups. E) Variance of POU5F1 and PAX6 in each of the sample groups. Significantly higher variance can be seen in the noninduced D2-8 group. F) Enrichment of forebrain and hindbrain development specific gene sets amongst the most significantly differentially expressed (DE) genes. Hypergeometric tests were run to test for enrichment (dashed lines) in either gene set amongst DE genes chosen from an ANOVA using the P value cut off indicated on the x-axis. Dotted lines indicate the percentage of each gene set amongst the DE genes at each cut off.
SC_12-0152_sm_supplFigure6.tif2849KSupplementary Figure 6 The expression profiles of the 14 differentially expressed genes, involved in nervous system development, at D2-8 of differentiation with LDN/SB versus LDN/Cpd2 and LDN/Cpd4.
SC_12-0152_sm_supplFigure7.pdf669KSupplementary Figure 7 Cells generated with LDN/SB can differentiate to neurons. Top left picture - neuronal differentiation by β-III-TUBULIN staining. Top right picture shows TYROSINE HYDROXYLASE (TH) staining characteristic for dopaminergic neurons. Bottom left – PERIPHERIN labeled PNS neurons are shown. Bottom right- GABAergic neurons.
SC_12-0152_sm_supplFigure8.pdf1027KSupplementary Figure 8 Single cell qPCR analysis show intermediate populations of cells coexisting during the time course of induction. A) First two principal components (PC1 and PC2) of a principal component analysis showing clustering of all cells. Cells are colored according to the time of collection. B) Mean cell-cell distance (defined as 1-R) for all cells collected at a given time point. Error bars indicate one standard error C) Expression heatmap of all cells grouped according to the clusters used in Figure 7, from cluster 1 (top) to cluster 7 (bottom).
SC_12-0152_sm_supplTable1.tif1793KSupplementary Table 1 TaqMan Gene expression assays list used in experiments.
SC_12-0152_sm_supplTable2.tif883KSupplementary Table 2 Supplementary Table with Kd values for tested compounds: Cpd2, Cpd3, Cpd4, Cpd7, SB, DM and LDN against ALK1/2/3/4/5/6, ACVR2A, ACVR2B, BMPR2, TGFBR2. The values were determined using 11 point dose response curse and are shown as average of technical duplicate.
SC_12-0152_sm_supplTable3.tif1939KSupplementary Table 3 GO biological process level 4 gene sets enriched (Benjamini & Hochberg P < 0.01) in genes differentially expressed between SB and SB-analogue induced samples.
SC_12-0152_sm_suppl_Data.pdf107KSupplementary Data

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