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. Author manuscript; available in PMC: 2022 Jun 3.
Published in final edited form as: Cell Stem Cell. 2021 Apr 28;28(6):1105–1124.e19. doi: 10.1016/j.stem.2021.03.005

Figure 2. PDLOs recapitulate cell type-specific features.

Figure 2.

(A) Representative overview IF images of HUES8-derived PDLOs. (B) Downregulation of PP and upregulation of ductal markers in PDLOs in qPCR experiments (PTF1A, PDX1, KRT7, CFTR: n=3; NKX6-1, ALB, SOX9, KRT19: n=6; in duplicates). (C) Representative IF images of individual PDLOs. (D) Time-resolved downregulation of PP markers measured by flow cytometry (FC) in comparison with patient-derived human PDAC organoids (Panc163) (n=4; d45/73: n=3; Panc163: n=2; in duplicates). (E) IF images of PDLOs stained for ductal, epithelial, proliferation, and polarity markers. (F) IF staining for CFTR, tight junction protein Occludin (OCLN), and primary cilia (acTUB, acetylated Tubulin). Scale bar: 10 μm. (G) Transmission electron microscopy images of a PDLO. Arrow marks a desmosome, dashed arrow microvilli. (H) Carbonic anhydrase (CA) activity assay (n=3; in duplicates, 3 blinded measurements for each replicate). Right: Higher CA2 level in PDLOs than in PPs on Western blot (WB) (n=2; in duplicates). (I) PDLO swelling within the CFTR assay upon stimulation with 20 μM forskolin (FSK) and 100 μM IBMX for 18 h (n=3; in duplicates). Right: BF images of PDLOs (d44/45). (J,K) Confirmation of the functional similarity in ion secretion of PDLOs and adult primary tissue-derived ductal organoids by intracellular pH measurement. (J) Apical Cl−/HCO3– exchange activity (PDLOs: n=28; Primary organoids: n=12) (Maléth et al., 2015) and (K) basolateral Na+ dependent HCO3− uptake (PDLOs: n=15; Primary organoids: n=13; n=number of organoids) (Molnár et al., 2020) were estimated (Mean±SEM; analysis of variance/Mann-Whitney test). (L,M) IF images of KRT19 and E-CAD in PDLOs derived from Co-iPSCs or H1 together with progenitor and ductal mRNA marker expression (n=3; in duplicates). Scale bars: 100 μm, if not stated elsewise. Insets in the corners are 4x enlarged. PDLOs represent day 30 of the protocol. B,L,M: Floating bars spanning minimal and maximal values; multiple t-tests via the Holm-Sidak method; only significant comparisons are depicted. D,H,I: Mean±SEM; ordinary one-way Anova followed by Tukey’s multiple comparison test.