Figure 2.

WNT3 Is a Functional Biomarker for Predicting the DE Differentiation Potential

(A) WNT3 levels in control and various knockdown HUES8 sublines correlate with their DE differentiation efficiency. Five shRNAs and one control shRNA were used to generate HUES8 sublines.

(B) Overexpression of WNT3 in the H9.2 hESC line in the undifferentiated state can improve the DE differentiation efficiency. The improvement correlates with the level of WNT3 expression.

(C) Independent verification of the predictive potential of WNT3 using different hPSC lines maintained in a different lab. hiPSC line JT16 and hESC lines HES3 (NKX2-5-GFP), H9, MEL-1 (NKX2-5-GFP), and H7 cultured on feeder or defined E8 system were used in this assay.

(D) qRT-PCR analysis of the expression of endoderm lineage genes and pluripotent genes after DE differentiation. Two cell lines, H7 and MEL-1, were chosen for this analysis based on their endogenous WNT3 expression levels in the undifferentiated state. Shown is one representative result (with SD from three technical replicates) of two independent experiments.

(E) Four previously described protocols (Jiang et al., 2013) were used to differentiate hESCs into DE, and the correlations between the WNT3 expression level of undifferentiated hESCs and the DE differentiation efficiency were analyzed. The four protocols included the following treatments in a serum-free medium: (1) A - > A (Activin A only for 4 days); (2) AW - > A (Activin A and Wnt3a for 2 days followed by Activin A only for another 2 days); (3) A - > AX (Activin A only for 2 days followed by Activin A and XAV 939 for another 2 days); and (4) AW - > AX (Activin A and Wnt3a for 2 days followed by Activin A and XAV 939 for another 2 days).

See also Figure S2.