To identify a molecular marker that can serve

To identify a molecular marker that can serve as an indicator of the differentiaion potential of the cell lines tested, we analyzed the lgk974 levels of a group of genes in hESCs that include pluripotent transcription factors, modulators of various signaling pathways known to be involved in DE differentiation (Mfopou et al., 2010), and epigenetic factors implicated in DE differentiation (Jiang et al., 2013). Among the genes analyzed, the expression level of WNT3 in hESCs has the best correlation with the DE differentiation potential (Figures 1B, 1C, and S1C), suggesting that the WNT3 expression level in hESCs is a potential biomarker for predicting the DE differentiation potential. If the WNT3 expression level in hESCs is a true predictor of the potential for DE differentiation, we anticipated that manipulation of the WNT3 level in hESCs should affect their DE differentiation capacity. To test for this possibility, we designed five different small hairpin RNAs (shRNAs) that target different regions of WNT3 and generated five stable WNT3 knockdown HUES8 sublines. We first confirmed that knockdown of WNT3 does not affect hESC maintenance, as the expression of the pluripotent factors was maintained in these WNT3 knockdown HUES8 sublines (Figure S2A). We then subjected the parental HUES8 line and derived sublines to the same differentiation protocol and determined their DE differentiation efficiency, which ranged from 10% to 80%, by quantifying the percentage of CD184 and CD117 double-positive cells. Interestingly, when the differentiation efficiency of these HUES8 sublines was plotted against the WNT3 levels in the undifferentiated cell state, we observed an excellent correlation (Figure 2A). Next, we asked whether the differentiation potential of a “poor” hESC line with a low DE differentiation potential could be improved by enforced expression of WNT3. To this end, we constructed a doxycycline-inducible WNT3 overexpression lentiviral vector and transfected it into H9.2, one of the hESC lines with a low endogenous WNT3 level and low DE differentiation potential (Figure 1). We first confirmed that doxycycline is able to induce WNT3 expression in this modified H9.2 hESC line in a doxycycline dosage-dependent manner (Figure S2B). We then confirmed that doxycycline treatment does not alter the expression of pluripotent genes (OCT4, NANOG, and SOX2) or AXIN2, a marker of canonical WNT/beta-catenin activity (Figure S2B). This indicates that transient overexpression of WNT3 alone does not affect hESC maintenance, and that WNT3 is functionally different from WNT signaling pathway activation, which could induce hESCs to differentiate (Davidson et al., 2012). After 4 days of culturing under different concentrations of doxycycline, these cells were subjected to DE differentiation (without doxycycline). We found that doxycycline-induced WNT3 expression in undifferentiated H9.2 cells improved their differentiation efficiency, and, more importantly, the improvement occurred in a WNT3 dosage-dependent manner (Figure 2B). Collectively, the above results support our observation that the DE differentiation potential correlates with WNT3 expression levels in hESCs. To evaluate the predictive potential of WNT3, we performed the same differentiation experiment at Duke University using all of the hESC/hiPSC lines available in the Bursac lab (including hESC lines HES3 [NKX2-5-GFP], H7, and H9 and MEL-1 [NKX2-5-GFP], and hiPSC line JT16). We first analyzed the WNT3 expression level in these cell lines by quantitative PCR (qRT-PCR), which allowed us to rank order these cell lines based on their WNT3 expression levels. We then subjected these cell lines to differentiation and quantified their differentiation efficiencies. In almost perfect agreement with our prediction, we found that the differentiation efficiency is highly correlated with the WNT3 levels of these hESC/hiPSC lines (Figure 2C), confirming the predictive capacity of WNT3. To further support our cell-surface-marker-based fluorescence-activated cell sorting (FACS) results, we analyzed the expression of endoderm lineage markers and pluripotent genes after DE differentiation. We chose two hESC lines (H7 [low WNT3] and MEL-1 [high WNT3]) based on their endogenous WNT3 expression levels. The results shown in Figure 2D clearly demonstrate that the expression levels of a panel of DE marker genes are higher in MEL-1 compared with the H7 cell line, suggesting a correlation between their DE differentiation potential and the endoderm marker gene-expression levels in hESCs.