1. Developmental Biology
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Reconstructing human pancreatic differentiation by mapping specific cell populations during development

  1. Cyrille Ramond
  2. Nicolas Glaser
  3. Claire Berthault
  4. Jacqueline Ameri
  5. Jeannette Schlichting Kirkegaard
  6. Mattias Hansson
  7. Christian Honoré
  8. Henrik Semb
  9. Raphaël Scharfmann  Is a corresponding author
  1. INSERM U1016, Cochin Institute, France
  2. CNRS UMR 8104, France
  3. University of Paris Descartes, France
  4. Unit for Lymphopoiesisis, France
  5. Faculty of Health Sciences, University of Copenhagen, Europe
  6. Novo Nordisk A/S, Europe
  7. Global Research External Affairs, Novo Nordisk A/S, Europe
Research Article
Cite this article as: eLife 2017;6:e27564 doi: 10.7554/eLife.27564
7 figures and 1 additional file

Figures

EPCAM expression in the human fetal pancreas.

(A) The flow cytometry plot represents CD45 and CD31 expression against EPCAM gated on live human fetal pancreatic cells (9.7WD), n = 9. (B) Immunohistochemistry for PDX1 and EPCAM on pancreatic section (9WD), n = 3. Scale bar = 100 μm. (C–E) Flow cytometry plots of PDX1 and NKX6-1 expression at 9.4WD on CD45+/CD31+ cells (red square), CD45-CD31-EPCAM- cells (TN = triple negative green square) and CD45-CD31-EPCAM+ cells (blue square). (F) RT-qPCR analysis of PDX1 and NKX6-1 expression on sorted CD45-CD31-EPCAM+ and TN cells. ND = Not Detected.

https://doi.org/10.7554/eLife.27564.002
Figure 2 with 2 supplements
GP2 and ECAD expression in the human fetal pancreatic epithelium.

GP2 and ECAD expressions were assayed by flow cytometry during development. (A) FACS plots display the expression at 9.4WD of CD45 and CD31 against EPCAM (left plot) and GP2 and ECAD gated on CD45-CD31-EPCAM+ (right plot). n = 4 (B) Cell frequencies of the GP2hi (GP2hiECAD+), GP2+ (GP2+ECAD+), GP2- (GP2-ECAD+) and Elow (GP2-ECADlow) populations at 9.4WD. n = 4 (mean ±SEM) (C) GP2 and ECAD expressions on fetal pancreases at 7-12WD gated on CD45-CD31-EPCAM+ cells. 7WD n = 2, 8.4WD n = 9, 9.4WD n = 4, 12WD n = 5. (mean ±SEM) (D) Cell frequencies of the GP2hi, GP2+, GP2- and Elow populations at 7-12WD. Cell frequencies were calculated from three independent experiments for each time point. (E) Scheme that represents the development of GP2hi, GP2+, GP2- and Elow populations.

https://doi.org/10.7554/eLife.27564.003
Figure 2—source data 1

Cell frequency at 9.4WD by flow cytometry.

Cell frequencies of the GP2hi (GP2hiECAD+), GP2+ (GP2+ECAD+), GP2- (GP2-ECAD+) and Elow (GP2-ECADlow) populations at 9.4WD. n = 4 (mean ± SEM). Cell frequencies were calculated from four independent experiments for each time point.

https://doi.org/10.7554/eLife.27564.004
Figure 2—source data 2

Cell frequency during development.

Cell frequencies of the GP2hi, GP2+, GP2- and Elow populations at 7-12WD. Cell frequencies were calculated from three independent experiments for each time point.

https://doi.org/10.7554/eLife.27564.005
Figure 2—figure supplement 1
Gating strategy for GP2 and ECAD.

Human fetal pancreas at 9WD was stained for CD45, CD31, EPCAM, ECAD and GP2. Doublets cells were excluded from the analysis with FSC-H and FSC-W (middle top plot). Propidium iodide was used to exclude dead cells as shown in the right top plot in the diagonal. GP2 and ECAD expressions were analyzed in CD45+/CD31+ (red square), CD45-CD31-EPCAM- (green square) and CD45-CD31-EPCAM+ (green square) compartments. CD45-CD31-EPCAM- population was used as negative control to set up the GP2-ECAD+ (named GP2-) and GP2+ECAD+ (named GP2+) gates. GP2+ECAD+ population was used to set up the gate for ECAD levels. This experiment is representative of 5 independent stainings at 9WD. This gating strategy was applied to each pancreatic stage.

https://doi.org/10.7554/eLife.27564.006
Figure 2—figure supplement 2
Expression of ECAD and GP2 during development.

(A) GP2 and ECAD expression by flow cytometry on CD45-CD45-EPCAM+ population at 8.4, 8.6 and 9.4WD. (B) ECAD expression in the GP2+ (in blue) and all GP2- (GP2-ECAD++GP2ECADlow, in red) populations at 8.4, 8.6 and 9.4WD. (C) GP2 expression by flow cytometry on CD45-CD31-EPCAM+ at 8.4, 10.6 and 13.1WD. GP2hi gates were fixed at 13.1WD for the three stages. (A, B) 8.4WD n = 7, 8.6WD n = 7 and 9.4WD n = 3. (C, D) 8.4WD n = 7, 10.6WD n = 3, 13.1WD n = 2.

https://doi.org/10.7554/eLife.27564.007
Figure 3 with 3 supplements
Transcriptomic analysis of the GP2hi, GP2+, GP2- and Elow populations.

(A) PCA map of sorted pancreatic cells (epithelium and mesenchyme). (B) Top enriched biological processes in each cell population compared to the mesenchyme. Results were obtained with GSEA software using the GO database. (C) PCA map of epithelial-sorted cells (GP2hi, GP2+, GP2- and Elow populations) from 9 (top map) and 11WD (bottom map). The number of specifically genes enriched in each population in each population (p<0.05) is displayed. PCA maps are displayed in 2D with the three Principal Components on figure Figure 3—figure supplement 1) and the Gene Ontology lists in Supplementary file 1a.

https://doi.org/10.7554/eLife.27564.008
Figure 3—figure supplement 1
Principal component analysis.

(A) 2D PCA maps of sorted pancreatic cells (epithelium and mesenchyme) displaying the first three Principal Components (PC1 against PC2, PC1 against PC3 and PC2 against PC3) from Figure 3A. (B) 2D PCA maps of sorted epithelial cells (GP2hi, GP2+, GP2- and Elow populations) at 9WD displaying the first three Principal Components (PC1 against PC2, PC1 against PC3 and PC2 against PC3) from Figure 3C (top map). (C) 2D PCA maps of sorted epithelial cells (GP2hi, GP2+, GP2- and Elow populations) at 11WD displaying the first three Principal Components (PC1 against PC2, PC1 against PC3 and PC2 against PC3) from Figure 3C (top map) from Figure 3C (bottom map).

https://doi.org/10.7554/eLife.27564.009
Figure 3—figure supplement 2
Heatmaps of genes from GO Biological processes digestion, insulin secretion and pancreas development.

Enriched genes from GO biological processes « digestion » (A), « insulin secretion » and «pancreas development» (B) were used to generate heatmaps. They display expression of these genes at 9 and 11WD in the GP2hi, GP2+, GP2-, Elow and Mesenchyme populations. Heatmaps were generated by the ‘heatmap2’ function from gplots R package on standardized log2 expression values, with Pearson correlation as the distance function.

https://doi.org/10.7554/eLife.27564.010
Figure 3—figure supplement 3
The GP2hi and Elow populations are enriched in acinar and endocrine markers respectively.

Comparative analyses using data from Segerstolpe et al. (2016) indicate that (A) Genes found enriched in the GP2hi population, are preferentially expressed in the human adult pancreas in acinar cells (B) Genes found enriched in the Elow population, are preferentially expressed in the human adult pancreas in endocrine cells. Heatmaps were obtained with Log2 RPKM values of the 1000 characterized single cells from (Segerstolpe et al., 2016).

https://doi.org/10.7554/eLife.27564.011
Figure 4 with 1 supplement
Characterization of the GP2hi, GP2+, GP2- and Elow populations.

(A,B) Expression of acinar (A) and endocrine (B) markers in the GP2hi, GP2+, GP2- and Elow populations by global transcriptomic analyses and by RT-qPCR. (C) Expression of NEUROG3 by RT-qPCR in the GP2hi, GP2+, GP2- and M populations. Heat maps and RT-qPCR are representative of 3 independents experiments. ND = Non Detected. M = CD45-CD31-EPCAM-. *p<0.05, **p<0.001, t test. (mean ± SEM).

https://doi.org/10.7554/eLife.27564.012
Figure 4—figure supplement 1
Expression of ductal markers in the GP2hi, GP2+, GP2- and Elow populations.

Expression of ductal markers in the GP2hi, GP2+, GP2- and Elow populations by transcriptomic analysis (A) and RT-qPCR (9-13WD) (B). ND = Not Detected. M = CD45CD31-EPCAM-. Heatmaps and RT-qPCR are representative of 3 independents experiments. (mean ± SEM).

https://doi.org/10.7554/eLife.27564.013
Figure 5 with 1 supplement
Expression of CD142 and SUSD2 in the GP2hi, GP2+, GP2- and Elow populations.

(A) Expression of CD142 and SUSD2 in GP2+, GP2- and Elow populations at 9WD by microarray analysis. Boxplots were obtained using standardized log2 expression values. (B) Expression of CD142 and SUSD2 in the GP2hi (in green), GP2+ (in red), GP2- (in purple) and Elow (in orange) populations at 9.4WD by flow cytometry. GP2 and ECAD expressions were gated on live CD45-CD31-EPCAM+ cells. (C) Expression of CD142 and SUSD2 in the GP2+ (left plot), GP2- (right plot) and Elow (far right plot) populations at 7WD, 8.4WD, and 11.3WD. (D) Cell frequencies of the GP2-CD142+SUSD2- (named CD142+SUSD2- in yellow) and the GP2-CD142-SUSD2- (CD142-SUSD2- in red) subsets from 7WD to 9.4WD. (mean ± SEM). (E) Cell frequencies of the ElowGP2-CD142-SUSD2+ (named ElowSUSD2+, in purple) and the ElowGP2-CD142-SUSD2- (ElowSUSD2-, in green) subsets from 7 to 12WD. (mean ± SEM). (A) n = 2–3, (B) n = 3; C) 7WD n = 2, 8.4WD to 9.4WD n = 3. (D) n = 3.

https://doi.org/10.7554/eLife.27564.014
Figure 5—figure supplement 1
Expression of CD142 and SUSD2 in the GP2+, GP2- and Elow populations at 8.6WD.

Expression of CD142 and SUSD2 in the GP2+ (in red), GP2- (in purple) and Elow (in orange) populations at 8.6WD by flow cytometry. GP2 and ECAD expressions were gated on live CD45-CD31-EPCAM+ cells. n = 7.

https://doi.org/10.7554/eLife.27564.015
Figure 6 with 1 supplement
Molecular characterization of the GP2-CD142-SUSD2- and ElowGP2-CD142-SUSD2- subsets.

(A, B) RT-qPCR for CHGA, NEUROD1, NKX2-2 and INS on sorted GP2-CD142+SUSD2- (GP2-CD142+), GP2-CD142-SUSD2- (GP2-CD142-), ElowGP2-CD142-SUSD2+ (ElowSUSD2+) and ElowGP2-CD142-SUSD2- (ElowSUSD2-) subsets at 8.6 and 10-12WD. (C) RT-qPCR for NEUROG3 at 8.4, 8.6, 9 and 11-13WD in the GP2-CD142+SUSD2- (GP2-CD142+), GP2-CD142-SUSD2- (GP2-CD142-), ElowGP2-CD142-SUSD2+ (ElowSUSD2+) and ElowGP2-CD142-SUSD2- (ElowSUSD2-) subsets. (D, E) Single cell RT-qPCR at 9WD on 172 GP2-CD142+SUSD2- (GP2-SUSD2-, left panel) and 166 ElowGP2-CD142-SUSD2+ (SUSD2+, right panel) cells for the expression of PPIA, NEUROG3, NEUROD1, NKX2-2 and CFTR. Pie charts represent the percentage of NEUROG3+ (in orange), NEUROG3-CFTR+ (in blue) and NEUROG3-CFTR- cells (in grey). For NEUROG3+ the percentages of NEUROD1+, NKX2-2+, and CFTR+ cells are displayed. Each line represents one cell. PPIA- cells were excluded from the analysis. (A–C) n = 3, (D, E) n = 2. *p<0.05, ****p<0.0001, t test. (mean ±SEM).

https://doi.org/10.7554/eLife.27564.016
Figure 6—figure supplement 1
Expression of CHGA and NEUROG3 in the ElowCD142+ subsets.

RT-qPCR for CHGA and NEUROG3 in the GP2-CD142+SUSD2- (named GP2-CD142+), ElowCD142+, ElowGP2-CD142-SUSD2+ (named ElowSUSD2+) and M (CD45-CD45-EPCAM-) populations. Heatmaps and RT-qPCR are representative of 3 independents experiments. (mean ± SEM).

https://doi.org/10.7554/eLife.27564.017
Figure 7 with 1 supplement
Expression of CD142, ECAD and SUSD2 in pancreatic endocrine cells derived from hPSCs.

(A) Scheme of in vitro differentiation of hPSCs into endocrine pancreatic cells. (B, C) Expression of CD142, ECAD (gated on lived CD45-CD31-EPCAM+ cells for the human) and SUSD2 in CD142+, CD142- and Elow population by flow cytometry in the human fetal pancreas at 9.4WD (in B) and in pancreatic endocrine cells derived from hPSCs (AD3.1 iPSC) at stage 5 (in C). (D) RT-qPCR on CD142+E-CAD+SUSD2- (named CD142+), CD142-E-CAD+SUSD2- (named CD142-), CD142-E-CADlowSUSD2+ (named ElowSUSD2+), CD142-E-CADlowSUSD2- (named ElowSUSD2-) from hPSCs (AD3.1 iPSC) at stage five for NEUROG3, NEUROD1 and NKX2-2). (E) Cell frequencies of CD142+SUSD2- (in yellow) and CD142-SUSD2- (in red) from hPSCs (SA121 hESC, AD2.1 iPSC, AD3.1 iPSC) from stage 2–5. (F) Cell frequencies of ElowSUSD2+ and ElowSUSD2- from hPSCs (SA121 hESC, AD2.1 iPSC, AD3.1 iPSC) from stage 2–5. (G) Scheme representing human pancreatic differentiation across development using cell surface markers. (B–F) n = 3. *p<0.05, **p<0.001, ***p<0.005, t test.

https://doi.org/10.7554/eLife.27564.018
Figure 7—figure supplement 1
Expression of CD142 and SUSD2 in the mesenchyme, the endothelial/hematopoietic and the epithelial compartments.

Human fetal pancreas at 10.6WD was stained for CD45, CD31, EPCAM, CD142 and SUSD2. CD142 and SUSD2 expressions are displayed in the CD45+/CD31+ (in red), CD45-CD31-EPCAM- (in green) and in CD45-CD31-EPCAM+ (in blue) populations on lived cells. n = 5.

https://doi.org/10.7554/eLife.27564.019

Additional files

Supplementary file 1

Gene Ontology list.

(a) List of gene sets and corresponding genes used in GSEA analysis displayed in Figure 3B. Gene sets were obtained from the gene ontology database. (b) List of differentially expressed genes specific to each population at 9WD. Elow: in yellow, GP2-: in purple, GP2+: in red, M: white. Values are displayed in Log2. (c) List of differentially expressed genes specific to each population at 11WD. Elow: in yellow, GP2-: in purple, GP2+: in red, M: white. Values are displayed in Log2.

https://doi.org/10.7554/eLife.27564.020

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