Figures and data
Regulated genes and signaling pathways during PP expansion under initial conditions
(A) Regression lines of two samples showing exponential expansion of PP cells for 32 d using initial conditions (CINI).
(B) MDS plot representing the Euclidian distance of the samples p0 (n=4), p5 (n=3) and p10 (n=3).
(C) Enrichment plots for regulated genes (p0 vs P10) of the TGF-ß signaling pathway, E2F target genes and DNA replication showing a negative correlation of the TGF-ß pathway with the expansion but a positive correlation of E2F target genes and DNA replication with the expansion. (D-H) Average transcript levels in normalized RNA-Seq counts of p0 (n=4), p5 (n=3) and p10 (n=3) cell populations for genes encoding signals or receptors of the TGFβ (D), FGF (E) and PDGF (F) signaling, the RA producing enzyme ALDH1A1 (G) as well as components of NOTCH (H) signaling.
Horizontal lines represent the mean ± SD. Statistical tests were one-way ANOVA using p0 as the control condition for the comparison with p ≤ 0.033 (*), p ≤ 0.002 (**), ≤ 0.0002 (***) and ≤ 0.0001 (****).
Reproducible expansion of PP cells in condition 5
(A) Growth curves and regression analysis for PP cells expanded in C0, C1 and C5 for at least 10 passages. The doubling time (Td) of C5-expanded cells (n=7) was 2.3 days with a 95% confidence interval (CI) of 2.13-2.51 days. This was clearly increased compared to C0-(n=2, Td=3.92 days, 95% CI = 3.22-4.98 days) and C1-expanded cells (n=2, Td=3.55 days, 95% CI = 2.88-4.62 days). The translucent shading represents the 95% CI of the growth rate of the different conditions.
(B-D) Gene expression profile of C0-, C1- and C5-expanded cells as shown by qPCR for expression of the key pancreas progenitor markers PDX1 (B), NKX6.1 (C) and SOX9 (D) during the expansion. Expression is normalized against expression of each marker at p0.
(E-G) Representative images of immunofluorescent staining of p0 PP cells (E) as well as C5-expanded cells at p5 (F) and p10 (G) for the PP transcription factors PDX1, NKX6.1 and SOX9. (H-J) Flow cytometry analysis of p0 PP cells (H), as C5-expanded cells at p5 (I) and P10 (J) for PDX1, NKX6.1 and SOX9.
(K) Cumulative results of the flow cytometry analyses for PDX1+/SOX9+ and PDX1+/SOX9+/NKX6.1+ C5-expanded cells at p0, p5 and p10.
Horizontal lines represent the mean ± SD. Statistical tests were two-way ANOVA with Tukey’s test, using p0 as the control condition for the comparison with p ≤ 0.033 (*), p ≤ 0.002 (**), ≤ 0.0002 (***) and ≤ 0.0001 (****). Scale bar corresponds to 50 um.
PP expansion conditions promote primarily proliferation rather than survival of PP cells.
(A, B) Histogram plots showing the % of cells that had incorporated EdU-Alexa488 in the CINI-expanded PP cells (A) in comparison to the C5-expanded PP cells (B).
(C) Summary of flow cytometry data comparing proliferation using EdU-Alexa488 incorporation (n=4) as well as cell death by Annexin V/7-AAD staining in INI- and C5-expanded PP cells (n=6).
Horizontal lines represent mean ± SD. Means were compared with multiple t-tests and significance is p ≤ 0.033 (*), p ≤ 0.0021 (**), p ≤ 0.0002 (***) or p ≤ 0.0001 (****).
Reproducible expansion in condition 6 promotes PP identity
(A) Growth curves and regression analysis for PP cells expanded in C5- and C6 for ten passages. The regression line for C5 showed a doubling time of 2.3 d (n=7) compared to C6 with 2.5 d (n=10).
(B-D) Representative images of immunofluorescent staining of p0 PP cells (B) as well as C6-expanded cells at p5 (C) and p10 (D) for the PP transcription factors PDX1, NKX6.1 and SOX9. (E-G) Flow cytometry analysis of non-expanded p0 PP cells (E) and C6-expanded cells at p5 (F) and p10 for PDX1+/SOX9+/ NKX6.1+ cells (G).
(H, I) Cumulative results of the flow cytometry analyses for PDX1+/SOX9+ and PDX1+/SOX9+/NKX6.1+ C6-expanded cells at p0, p5 and p10 (H) and comparison of the % of C5- and C6-expanded PDX1+/SOX9+/NKX6.1+ cells at p5 and p10 (I).
(J) Karyotyping of C6-expanded PP cells after sixteen passages showed no chromosomal abnormalities.
Horizontal lines represent the mean ± SD. Statistical tests were two-way ANOVA with Tukey’s test, using p0 as the control condition for the comparison with p ≤ 0.033 (*), p ≤ 0.002 (**), ≤ 0.0002 (***) and ≤ 0.0001 (****). Scale bar corresponds to 50 um.
Expansion stabilizes PP cell identity by repressing endocrine differentiation.
(A, B) Correlation analyses of the transcriptome profiles of non-expanded (p0) and p10 expanded PP cells (A) and the transcriptome profiles of p5 and p10 expanded cells (B). The numbers of upregulated and downregulated genes (normalized counts ≥ 200 and 0.5 ≥ FC ≥ 2) are shown in red and blue, respectively and r is the correlation coefficient.
(C) Most affected biological processes between p0 and p10.
(D) PCA of feeder expanded cells and corresponding p0 cells (shades of green), fibronectin (FN) expanded cells and corresponding p0 cells (shades of blue) as well as vitronectin-N (VTN-N) expanded cells and corresponding p0 cells (shades of red). Darker shades correspond to earlier passages.
(E) Expression levels of GP2 in normalized RNA-Seq counts.
(F) GP2 immunofluorescence of p12 expanded PP cells.
(G) Expression levels of progenitor and endocrine markers in normalized RNA-Seq counts.
Expansion of H9-derived and CRTD1-derived PP cells
(A, B) Growth curve and regression analysis of the expansion of H9-derived PP cells (A) and CRTD1-derived PPs cells (B).
(C, D) Flow cytometry analysis for PDX1+/SOX9+ and PDX1+/SOX9+/NKX6.1+ cells during the expansion in C6 of H9-derived PP cells (C) and CRTD1-derived PP cells (D) at p0 and during their expansion in C6 at p5 and p10.
Differentiation of ePP cells into SC-islets containing functional β-cells.
(A, B) Immunofluorescence analysis of SC-islets derived from p0 PP cells (dPP) or expanded PP cells for at least ten passages (ePP) for INS and GCG expression (A) or INS and SST expression (B).
(C) Percentages of INS+, INS+/GCG+ as well as GCG+ cells in SC-islets derived from dPP or ePP cells as determined by flow cytometry.
(D) Expression levels of NKX6.1, PDX1 and SLC30A8 in relation to expression levels in hPS cells (fold induction) as determined by qPCR.
(E) Secretion of C-peptide following sequential stimulation by 16.7 mM glucose and 16.7 mM glucose / 30 mM KCl after exposure in basal conditions with 2.8 mM glucose. Stimulation index is determined by the ratio of secretion under these conditions to secretion in basal conditions.
Horizontal lines represent the mean ± SD. Statistical tests were two-way ANOVA with Tukey’s test, using p0 as the control condition for the comparison with p ≤ 0.033 (*), p ≤ 0.002 (**), ≤ 0.0002 (***) and ≤ 0.0001 (****). Scale bar corresponds to 50 um. Scale bar corresponds to 100 μm (E, F)
