Figure 2
Characterization of Gibco and XCL-1 hiPSCs Undergoing LPM Differentiation by High Content Imaging and qRT-PCR. (A,B) High content imaging-based 5-color assay of Gibco hiPSCs cultured in mTeSR1 (A) versus APEL2 + GSKi (B) for 2 days. hiPSCs were co-stained for DAPI (panel 1), FOXF1 (panel 2), SALL4 (panel 3), OCT4 (panel 4), and T (panel 5). Panel 6 contains a false-colored merged image of OCT4 (red), T (green), FOXF1 (white) and SALL4 (blue) expression. (C) Quantification of marker expression in Gibco hiPSCs by high content imaging, presented as the mean ± SEM aggregated from 3 independent experiments of the % nuclei expressing each marker. Thresholding for each marker was performed relative to a non-expressing control cell type (human endothelial cells) for SALL4 and OCT4 or relative to undifferentiated hiPSCs for FOXF1 and T. (D,E) Confocal laser scanning microscopy images of Gibco hiPSCs cultured in mTeSR1 (D) versus APEL2 + GSKi (E) conditions for 2d and co-stained for DAPI (panel 1), FOXF1 (panel 2), and NANOG (panel 3). (F–J) Scatter plot of FOXF1 and NANOG intensity of DAPI + objects tabulated from cultures of Gibco hiPSCs (F), XCL-1 CRBN Mock hiPSCs (G), XCL-1 CRBN KO/KI hiPSCs (H), XCL-1 SALL4 Mock hiPSCs (I), and XCL-1 SALL4G416A hiPSCs (J) that were cultured either in mTeSR1 (black circles) or APEL2 + GSKi (red circles) and co-stained for DAPI, FOXF1 (x-axis), and NANOG (y-axis). (K) qRT-PCR characterization of Gibco hiPSCs and XCL-1 hiPSCs cultured in APEL2 + GSKi for 2 days relative to the undifferentiated control for each cell type cultured in mTeSR1. Data are presented as the log2 fold change relative to ACTB and undifferentiated control for pluripotency markers NANOG, POU5F1, and SALL4, primitive streak genes TBXT and SOX17, LPM genes FOXF1, GATA4, and KDR, and limb mesoderm genes PITX1 and TBX5. Color scales denote increased expression (red) or decreased expression (blue) relative to undifferentiated control.
