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

sc-12-0191_sm_SupplData.pdf17KSupplementary Methods
sc-12-0191_sm_SupplFigure1.pdf744KSupplementary Figure 1: NKX2.1 quantification of Condition A at day 11 by FACS. (A-B) The average proportion of NKX2.1 positive cells in Condition A was 75.18% ± 7.9 SEM (n=3). FACS analysis plots of NKX2.1 were set against the side scatter and gates set according to hESCs expression levels. (C-F) Low and high power image of NKX2.1 staining of Condition A at day 11. Scale bars: C-D = 500μm and E-F =100μm.
sc-12-0191_sm_SupplFigure2.tif107KSupplementary Figure 2: QPCR analysis of GLI1, GLI2 and GLI3 in Condition A at day 4. (A-C) Analysis of levels were compared to hESCs and the fold change levels for hESC set at 1. GLI1, GLI2 and GLI3 all showed significantly higher level in Condition A at day 4 (P < 0.05). Star indicates significance (P < 0.05).
sc-12-0191_sm_SupplFigure3.tif970KSupplementary Figure 3: FACS analysis of Condition CEarly (Day 4) for SOX2 and OCT4. (A-B) FACS analysis was performed and gates set according to hESCs for SOX2 and OCT4 expression. The average proportions of SOX2 and OCT4 positive cells for Condition CEarly were 89.17% ± 2.7 SEM and 0.91% ± 0.12 SEM (n=3), respectively.
sc-12-0191_sm_SupplFigure4.tif994KSupplementary Figure 4: FACS analysis of Conditions D-G for FOXA2. FACS analysis was performed and gates set according to hESCs. FACS analysis of Conditions D, F and G all yielded high percentages of FOXA2 positive cells, 86% (±2.8 SEM), 83.5% (±5.9 SEM) and 85.7% (±5.7 SEM) respectively. Condition E resulted in few (3.3%; ±1.2 SEM) FOXA2 positive cells, significantly lower than the other conditions (P < 0.0001). Sample size is n=3 for each condition.
sc-12-0191_sm_SupplFigure5.tif83KSupplementary Figure 5: QPCR analysis for mesoderm and endoderm markers in Condition D and E at day11. (A-B) BRACHYURY and SOX17 expression levels were analysed in Conditions D and E and compared to hESC with the fold change levels for hESC set at 1. No significant differences in expression levels were found.
sc-12-0191_sm_SupplFigure6.pdf1859KSupplementary Figure 6: Forebrain marker expression in Condition E at day 11. (AD) FOXG1 positive cells were detected throughout Condition E at day 11 and none were detected in Condition D. (E-H) OTX2+/PAX6+ cells were detected in Condition E at day 11. A-D Scale bar: 50μm

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