Fig 2: Efficient differentiation of XM001 iPSCs into pancreatic progenitors. (A) Schematic of iPSC-derived pancreatic progenitor differentiation protocol. (B) Immunostaining for FOXA2 and SOX17 on day 3. Scale bar indicates 50 µm. (C) Representative FACS plot of SOX17+ cells at DE stage. A differentiated sample stained with only the secondary antibody and XM001 iPSCs were used as negative controls. (D) FACS quantification of the percentage of SOX17+ cells at DE stage (n = 3). (E) Immunostaining for PDX1 on day 10. Scale bar indicates 50 µm. (F) Representative FACS plot of PDX1+ cells at the PP stage. A differentiated sample stained with only the secondary antibody and XM001 iPSCs were used as negative controls. (G) FACS quantification of the percentage of PDX1+ cells at PP stage (n = 3).

Fig 3: Crotonate promotes endoderm differentiation in the STEMDIFF endoderm differentiation medium(A) Crotonate dose-dependently promotes the expression of endodermal genes after endoderm differentiation of mel1 hESCs in the STEMDIFF endoderm differentiation medium. 4 × 104 mel1 cells/well (1.05 × 104/cm2) of a 12-well plate was induced with 1 mL (0.26 mL/cm2) STEMDIFF endoderm differentiation medium in the absence or presence of 5 mM crotonate for 48 h. RNA was collected, and the expression of the indicated genes was analyzed by qPCR with GAPDH as a loading control (n = 3 biological replicates/group, *p < 0.05, values are expressed as mean ± SEM). Figure reprinted with permission from Fang et al., 2021.(B) Crotonate promotes homogeneous endoderm differentiation of hESCs. 4 ×104 mel1 cells/well (1.05 × 104/cm2) of a 12-well plate were induced with 1 mL (0.26 mL/cm2) STEMDIFF endoderm differentiation medium with or without 5 mM crotonate for 3 days (~66 h). Cells were stained with anti-SOX17 and anti-FOXA2 antibodies, then subjected to flow cytometry analysis. Cells stained with secondary antibodies only were used as negative controls.(C) Crotonate promotes more hESCs to differentiate into endodermal cells in the STEMDIFF endoderm differentiation medium when analyzed at the single-cell transcriptome level. 82 × 104 mel1 cells/10-cm dish (1.05 ×104/cm2) were seeded and induced with 10 mL (0.13 mL/cm2) STEMDIFF endoderm differentiation medium with or without 5 mM crotonate for 3 days (~66 h). Cells were collected and processed for single-cell RNAseq. Compared to the differentiation condition in (B), only half amount of endoderm differentiation medium was used in this experiment when considering volume per area). Figure reprinted with permission from Fang et al., 2021.(D) Crotonate dose-dependently promotes the expression of endodermal genes after endoderm differentiation of H9 hESCs in the STEMDIFF endoderm differentiation medium. 8 × 104 H9 cells/well of a 12-well plate were induced with the STEMDIFF endoderm differentiation medium with or without crotonate for 3 days. RNA was collected, and the expression of the indicated genes was analyzed by qPCR with GAPDH as a loading control (n = 3 biological replicates/group, *p < 0.05, values are expressed as mean ± SEM). Figure reprinted with permission from Fang et al., 2021.

Fig 4: Endoderm differentiation of hESCs under different cell densities(A) Phase contrast images of mel1 hESCs seeded at 2 × 104, 4 × 104, 8 × 104, or 16 × 104/well of a 12-well plate before induction (Day 0), and at 3 days after induction (Day 3). Mel1 cells were seeded at the indicated density. On day 0, cells were washed with DPBS once, and incubated with 1 mL (0.26 mL/cm2) STEMDIFF endoderm differentiation medium together with 5 mM crotonate for 3 days. Scale bar: 400 µm.(B) High seeding cell density reduces endoderm differentiation. Mel1 cells were seeded at indicated densities and induced for endoderm differentiation for 3 days as described in (A). The total RNA was extracted, and the mRNA levels of SOX17 and FOXA2 were analyzed by qPCR and normalized with the mRNA levels of GAPDH (n = 3 biological replicates/group, *p<0.05, values are expressed as mean ± SEM).

Fig 5: Association of long intergenic non-coding RNA 261 (LINC00261) with epithelial–mesenchymal transition (EMT). (a) Gene set enrichment analysis (GSEA) of PDAC samples with low (n = 48) versus high LINC00261 expression (n = 48). Median LINC00261 expression was used as a cut-off; NES, normalized enrichment score; FDR, false discovery rate. (b) GSEA analysis of genes that were significantly inversely correlated with LINC00261 in the pancreatic adenocarcinoma (PDAC) dataset of Bailey et al. identified EMT as the highest enriched hallmark gene set. (c) Correlation of selected epithelial and mesenchymal marker genes with LINC00261 expression (* p < 0.05, ** p < 0.01, *** p < 0.001). Note: CDH1, E-cadherin; KRT19, keratin 19; CLDN7, claudin 7; CDS1, CDP-diacylglycerol synthase 1; SCNN1A, sodium channel epithelial 1 subunit alpha; FOXA2, forkhead box A2; CDH2, N-cadherin; VIM, vimentin; FN1, fibronectin 1; ZEB1, zinc finger E-box binding homeobox 1; ZEB2, zinc finger E-box binding homeobox 2; SNAI1, snail family transcriptional repressor 1; SNAI2, snail family transcriptional repressor 2; TGFB1, transforming growth factor beta 1; MMP2, matrix metallopeptidase 2; PLAUR, plasminogen activator, urokinase receptor; AXL, AXL receptor tyrosine kinase.