Download Supplementary Figure 1. Current definitive endoderm (DE

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Transcript
Supplementary Figure 1. Current definitive endoderm (DE) differentiation protocols are neither
efficient nor robust. A previously published DE protocol (8) applied to several hESC lines (H1, H9, HUES1,
HUES9) results in poor DE induction (n=3 biologically independent experiments; (mean± S.D.).
Supplementary Figure 2. Cell line specific effects of extracellular matrix proteins (ECMPs) in promoting
differentiation of hESCs to definitive endoderm (DE). (a) Dendrogram of similarity relationship between
biologically independent array experiments demonstrates the consistent effects of the ECMP
combinations within each hESC line. (b) The Pearson correlation coefficient matrix reveals the hESC linespecific effects of certain ECMP in promoting efficient DE differentiation.
Supplementary Figure 3. Definitive endoderm induction on extracellular matrix protein (ECMPs) in
additional hESC lines. Representative images of anti-SOX17 immunofluorescence of (a) HUES1 hESCs
and (b) H9 hESCs differentiated to definitive endoderm (DE) on Fibronectin (FN), Vitronectin (VTN), and
Fibronectin and Vitronectin (FN + VTN) (mean± S.E.M.). Gene expression analysis of endoderm genes
SOX17 and FOXA2 of (c) HUES1 hESCs and (d) H9 hESCs differentiated to definitive endoderm (DE) on
Fibronectin (FN), Vitronectin (VTN), and Fibronectin and Vitronectin (FN + VTN) (mean± S.E.M.).
Supplementary Figure 4. Expression of laminin (LN) integrin subunits in definitive endoderm (DE). (a)
Time course of laminin specific subunits ITGA6 (CD49f) during hESC (H1, H9, HUES1) to DE (n=3). (b)
Representative flow cytometry histograms of cell surface protein expression of laminin specific subunits
ITGA6 (CD49f) and ITGB1 (CD29) in hESCs and DE. (c) Quantification of percentage of cell surface protein
expression of ITGA6+ (CD49f+) and ITGB1+ (CD29+) hESCs and DE (HUES9 and HUES1; n=3; error bars,
S.E.M.).
Supplementary Figure 5. RFP and integrin expression in doxcycline (DOX) treated hESCs. (a) RFP
expression marks inducible shRNA expression (mean ± S.E.M.). (b-c) qPCR analysis of DOX treated
ITGA5shRNA and ITGAVshRNA hESCs. Analysis reveals that ITGAV (CD51) and ITGA5 (CD49e) gene expression
is unchanged in DOX treated ITGA5shRNA and ITGAVshRNA hESCs, respectively. (d) Wild-type HUES9 hESCs
were treated with DOX for 72 hrs. Quantitative PCR (qPCR) analysis shows DOX treatment alone does
not affect ITGA5 (CD49e) and ITGAV (CD51) gene expression. (n=3; error bars, S.E.M).
Supplementary Figure 6. Quantitative PCR (QPCR) analysis of hESC derived definitive endoderm (DE)
sorted for integrin α5 (ITGA5/CD49e) and integrin αv (ITGAV/CD51). Gene expression analysis of the
sorted populations reveals that double positive cells [ITGA5(CD49e)+ / ITGAV(CD51)+] expressed
significantly higher levels of SOX17 than single positive cells [ITGA5(CD49e)+ / ITGAV(CD51)- or
ITGA5(CD49e)- / ITGAV(CD51)+] or double negative cells (ITGA5(CD49e)- / ITGAV(CD51)-) (n=3; error
bars, S.E.M).
Supplementary Table 1. Raw data of heatmap generated in Figure 1d.
Supplementary Table 2. Antibodies used in this study.
Supplementary Table 3. Taqman gene expression assay primers used in this study.