Extended Data Fig. 9: Evaluation of iLSEP-specific siRNA knockdown.
From: Self-organization of sinusoidal vessels in pluripotent stem cell-derived human liver bud organoids
a) qRT-PCR analysis of WNT2, VEGFA, RSPO3, and F8. Data represent the mean ± SEM (n = 4 biological replicates; *, p < 0.05; multiple two-sided t-test with controlling FDR). In the “+Control,” siRNAs of non-targeted negative controls were used. b-c) Scatter dot-plot of the flow cytometry analysis of CD34/CD32 and CD73/CXCR4 expression in CD34+CD43- iLSEC with siRNA treatment at day14. d) Observation of fluorescent reporters with bright-field image to track GFP+ iLSEP and AlexaFlour555 (AF555)-tagged siRNA embedded in the same layer of multilayered gel. Scale bar indicates 500 µm. For at least 9 days, AF555-tagged siRNA remained in the packed gel or inside the organoids. e) Scatter dot-plot of the flow cytometry analysis of whole HLBO cells for separation of GFP−tagged iLSEP in second layer and GFP−negative cells in first layer in HLBO by FACSAria Fusion cell sorter. f) qRT-PCR analysis of WNT2 to validate GFP-tagged iLSEP specific knockdown efficiency. WNT2 mRNA expression levels were determined relative to the negative control siRNA treated for each GFP+ and GFP- fraction isolated. Data represent the mean ± SEM (n = 8, GFP+ groups; n = 7, GFP- in +Control; n = 6, GFP- in +siWNT2; all n are biological replicates; ***, p < 0.001; multiple two-sided t-test with Holm-Sidak correction). g) Cross-sectional H&E staining image of HLBOs with siRNA treatment. h) Measurement of VEGF-A in siRNA-treated HLBO supernatants at day3. Data represent the mean ± SEM (n = 4 biological replicates; ND, not detected). i) Whole-mount image of siRNA-treated HLBOs with immunofluorescent staining for CD31, STAB1 and LYVE1. j) Quantification of the percentage of cells expressing CD31 or LYVE1 alone or co-expressing CD31 and LYVE1. Data represent the mean ± SEM (n = 4 biological replicates; *, p < 0.05; **, p < 0.01; two-way ANOVA with Sidak’s multiple comparison test). k) Quantification of the percentage of STAB1 co-expressing endothelial cells (CD31+ or LYVE1+). Data represent the mean ± SEM (n = 4 biological replicates; ns, not significant; two-sided Welch’s t-test).
