Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

Abstract

The exocrine pancreas, consisting of ducts and acini, is the site of origin of pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Our understanding of the genesis and progression of human pancreatic diseases, including PDAC, is limited because of challenges in maintaining human acinar and ductal cells in culture. Here we report induction of human pluripotent stem cells toward pancreatic ductal and acinar organoids that recapitulate properties of the neonatal exocrine pancreas. Expression of the PDAC-associated oncogene GNAS^R201C induces cystic growth more effectively in ductal than acinar organoids, whereas KRAS^G12D is more effective in modeling cancer in vivo when expressed in acinar compared with ductal organoids. KRAS^G12D, but not GNAS^R201C, induces acinar-to-ductal metaplasia-like changes in culture and in vivo. We develop a renewable source of ductal and acinar organoids for modeling exocrine development and diseases and demonstrate lineage tropism and plasticity for oncogene action in the human pancreas.

Keywords: GNAS; KRAS; acini; cancer precursor; exocrine pancreas; lineage specification; organoid; pancreatic cancer; plasticity; pluripotent stem cell.

Graphical abstract

Figure 1

Commitment of human PP cells toward the acinar and ductal lineages (A) Schematic of the duct-like and acinus-like organoid induction protocols. (B) Phase-contrast images of organoids during 16-day culture (n = 3, independent cultures). Scale bar, 50 μm. (C) Changes in total area and form factor (circularity index) of organoids during morphogenesis (n > 150, n = 3 independent cultures). Box-and-whisker plot, range 5%–95%; center lines indicate median values; gray dots represent individual measurements. ^∗∗∗p < 0.001. (D) Immunostaining for collagen IV (red), DAPI (blue), and ZO1 (green). Scale bar, 50 μm. (E) TEM images of acinar and ductal organoids. Red arrow, electron-dense vesicles; N, nucleus; L, lumen. DU (blue), duct-like organoid; AC (orange), acinus-like organoid.

Figure 2

Single-cell analysis of hESC-derived duct-like and acinus-like organoids and expression of lineage markers and functional readouts (A) Uniform manifold approximation and projection (UMAP) of single-nucleus transcriptomes of cells in pancreatic progenitors (PPs; pink), DUs (blue) and ACs (orange). Shown are results from two independent cultures and sequencing. (B) Venn diagram of cell type markers identified by pairwise comparison using adjusted p ≤ 0.05 and average log(fold-change) > 0. The color scheme is the same as in (A). (C) Comparison of Pearson correlation coefficients for expression of marker genes (1,012 genes; adjusted (adj.) p < 0.05) between organoid culture and human neonatal acinar and ductal cells. Marker genes were derived by identifying differentially expressed genes (p < 0.05) in cells under different culture conditions. Wilcoxon rank-sum test was used. (D) Immunoblot analysis for acinar and ductal markers (n = 3 independent cultures). The numbers under the blots represent normalized signals of protein bands. (E and F) Morphological changes of day 8 DUs (blue) and ACs (orange) in response to 10 μM forskolin treatment (2-h incubation). Scale bar, 50 μm. (G) Measurements of organoid size changes induced by forskolin treatment. Box-and-whisker plot, range 5%–95%; center lines indicate median values; gray dots represent individual measurements (n > 200, combination of three independent cultures). (H–J) Measurement of enzyme activities in DU and AC lysates (n = 3 independent cultures). y axis, enzyme activity in arbitrary units. Error bars in bar charts represent standard deviation; gray dots represent individual measurements. ^∗∗∗p < 0.001; ^∗, 0.01 < p < 0.05; N.S., not significant, p > 0.05.

Figure 3

Temporal changes in marker expression during duct-like and acinus-like lineage specification (A–D) RNA expression of pancreatic ductal markers SOX9 (A), HNF1B (B), CAII (C), and CFTR (D) in DUs and ACs during 16-day culture. Floating column charts represent RNA measurements from quantitative PCR, hinges represent maximal and minimal values, central lines indicate mean values, and dots represent individual measurements. (E and F) Expression of SOX9 (E) and CAII (F) in organoids, detected by immunofluorescence. Numbers embedded in the images indicate the average percentage and standard deviation of marker-positive organoids. Scale bars, 50 μm. (G–I) RNA expression of the pancreatic acinar markers PTF1A (G), RBPJL (H), and CPA1 (I) in DUs and ACs during 16-day culture. Floating column charts: representation as in (A). (J and K) Expression of PTF1A (J) and CPA1 (K) in organoids, detected by immunofluorescence. Numbers embedded in images indicate the average percentage and standard deviation of marker-positive organoids. Scale bars, 50 μm. All results represent the sum of three independent cultures.

Figure 4

The GNAS mutant shows tropism for ductal-lineage epithelia (A) Immunoblot analysis for GNAS expression and phosphorylation of GNAS downstream effectors in DUs and ACs (n = 3, independent cultures). (B) Phase-contrast images of DUs and ACs expressing wild-type (WT) GNAS and GNAS^R201C. (-)DOX, without doxycline treatment; (+)DOX, with doxycycline treatment. Scale bar, 100 μm. (C) Change in size, measured as the total area of organoids expressing GNAS^WT or GNAS^R201C. Box-and-whisker plot, range 5%–95%; central lines, median values (n > 100, combination of three independent cultures). (D) Percentage of organoids surpassing the 99^th percentile of control organoid (no transgene expression) size (n = 3 independent cultures). (E) Organoid cell number changes induced by GNAS^WT and GNAS^R201C as detected by CellTiter. Bar charts represent the mean and standard deviation of measurements from three independent cultures; gray dots represent individual measurements. (F) Immunofluorescent staining of polarity proteins in organoids without (−DOX) and with (+DOX) GNAS^R201C expression. ZO1, red; collagen IV, green; DAPI, blue. Scale bar, 100 μm. (G) Expression of SOX9 in organoids without (−DOX) and with (+DOX) GNAS^R201C expression. SOX9, red; DAPI, blue. (H) AB (pH 2.5) staining of organoids without (−DOX) and with (+DOX) GNAS^R201C expression. Scale bar, 100 μm. (I) Expression of E-cadherin (green) and MUC2 (red) in DUs without (−DOX) and with (+DOX) GNAS^R201C expression. Scale bar, 50 μm. (J) Percentages of DUs expressing MUC2 (top chart) or MUC5AC (bottom chart). Bar charts represent the mean and standard deviation of measurements from three independent cultures; gray dots represent individual measurements. (K and L) RNA expression of lineage markers in DUs (K) and ACs (L) altered by GNAS^R01C expression. Floating column charts represent RNA measurements from quantitative PCR (n = 3 technical repeats), hinges represent maximal and minimal values, central lines indicate mean values, and dots represent individual measurements. ^∗∗∗p < 0.001; ^∗∗, 0.001 < p < 0.01; ^∗, 0. 01 < p < 0.05; N.S., p > 0.05.

Figure 5

KRAS^G12D induces ADM-like changes in ACs but a progenitor phenotype in DUs (A) Expression of the cell proliferation marker Ki67 (red) in DUs (blue) and ACs (orange) with and without KRAS^G12D expression. Scale bar, 50 μm. (B) Quantification of Ki67-positive organoids with and without KRAS^G12D expression. Box-and-whisker plot, range 5%–95%; center lines indicate median values; gray dots represent individual measurements. (C) Organoid cell number changes induced by KRAS^G12D expression. Bar charts represent the mean and standard deviation of measurements from three independent cultures; gray dots represent individual measurements. (D and E) Quantification of organoid morphological changes induced by KRAS^G12D expression. Bar chart representation as in (C). (F) Organoid size changes induced by KRAS^G12D expression. Box-and-whisker plot representation as in (B). (G and H) Expression of pancreatic lineage markers in DUs (G) and ACs (H) altered by KRAS^G12D expression. Floating column charts represent RNA measurements from quantitative PCR, hinges represent maximal and minimal values, central lines indicate mean values, and dots represent individual measurements. ^∗∗∗p < 0.001; ^∗∗, 0.001 < p < 0.01; ^∗, 0. 01 < p < 0.05; N.S., p > 0.05. All results represent the sum of three independent cultures.

Figure 6

TGF-β augments KRAS^G12D-induced phenotypes in ACs and DUs (A) Expression of the cell proliferation marker Ki67 (red) in DUs (blue) and ACs (orange) with KRAS^G12D expression and TGF-β treatment. Quantification of Ki67-positive organoids represented by box-and-whisker plots: range, 5%–95%; center lines indicate median values; gray dots represent individual measurement. (B and C) Organoid morphological changes induced by KRAS^G12D expression and TGF-β treatment. Bar charts represent the mean and standard deviation of measurements from three independent cultures; gray dots represent individual measurements. (D) Organoid size changes induced by KRAS^G12D expression and TGF-β treatment. Box-and-whisker plot representation as in (A). (E and F) Expression of pancreatic lineage markers in DUs (E) and ACs (F) altered by KRAS^G12D expression and TGF-β treatment. Floating column charts represent RNA measurements from quantitative PCR (n = 3), hinges represent maximal and minimal values, central lines indicate mean values, and dots represent individual measurements. (G–J) Detection of KRT7 (G), AB (H), MUC2 (I), and MUC5AC (J) in organoids under different experimental conditions. Bar chart representation as in (C). Scale bars in main images, 50 μm; scale bars in insets, 25 μm. ^∗∗∗p < 0.001; ^∗∗, 0.001 < p < 0.01; ^∗, 0. 01 < p < 0.05; N.S., p > 0.05. All results represent the sum of three independent cultures.

Figure 7

ACs expressing KRAS^G12D were more effective than DUs in inducing formation of early pancreatic-cancer-like lesions in vivo (A and B) Histology of lesions grown from KRAS^G12D-expressing DUs (A, N = 9) or ACs (B, N = 10) transplanted into the mouse pancreas. Tissues were stained with nuclear red. Scale bars, 100 μm. (C and D) Expression of human KRT19 (red), Ki67 (green), KRT7 (teal), MUC5AC (yellow), and SOX9 (purple) in lesions grown from KRAS^G12D-expressing DUs (C) or ACs (D). Scale bars, 100 μm.