Reconstructing human pancreatic differentiation by mapping specific cell populations during development

Abstract

Information remains scarce on human development compared to animal models. Here, we reconstructed human fetal pancreatic differentiation using cell surface markers. We demonstrate that at 7weeks of development, the glycoprotein 2 (GP2) marks a multipotent cell population that will differentiate into the acinar, ductal or endocrine lineages. Development towards the acinar lineage is paralleled by an increase in GP2 expression. Conversely, a subset of the GP2⁺ population undergoes endocrine differentiation by down-regulating GP2 and CD142 and turning on NEUROG3, a marker of endocrine differentiation. Endocrine maturation progresses by up-regulating SUSD2 and lowering ECAD levels. Finally, in vitro differentiation of pancreatic endocrine cells derived from human pluripotent stem cells mimics key in vivo events. Our work paves the way to extend our understanding of the origin of mature human pancreatic cell types and how such lineage decisions are regulated.

Keywords: development; developmental biology; human; pancreas; stem cells.

Figure 1.. 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. DOI: http://dx.doi.org/10.7554/eLife.27564.002

Figure 2.. 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 GP2^hi (GP2^hiECAD⁺), GP2⁺ (GP2⁺ECAD⁺), GP2^- (GP2^-ECAD⁺) and E^low (GP2^-ECAD^low) 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 GP2^hi, GP2⁺, GP2^- and E^low populations at 7-12WD. Cell frequencies were calculated from three independent experiments for each time point. (E) Scheme that represents the development of GP2^hi, GP2⁺, GP2^- and E^low populations. DOI: http://dx.doi.org/10.7554/eLife.27564.003

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. DOI: http://dx.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⁺+GP2ECAD^low, 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. GP2^hi 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. DOI: http://dx.doi.org/10.7554/eLife.27564.007

Figure 3.. Transcriptomic analysis of the GP2^hi, GP2⁺, GP2^- and E^low 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 (GP2^hi, GP2⁺, GP2^- and E^low 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. DOI: http://dx.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 (GP2^hi, GP2⁺, GP2^- and E^low 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 (GP2^hi, GP2⁺, GP2^- and E^low 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). DOI: http://dx.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 GP2^hi, GP2⁺, GP2^-, E^low 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. DOI: http://dx.doi.org/10.7554/eLife.27564.010

Figure 3—figure supplement 3.. The GP2^hi and E^low 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 GP2^hi population, are preferentially expressed in the human adult pancreas in acinar cells (B) Genes found enriched in the E^low 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). DOI: http://dx.doi.org/10.7554/eLife.27564.011

Figure 4.. Characterization of the GP2^hi, GP2⁺, GP2^- and E^low populations.

(A,B) Expression of acinar (A) and endocrine (B) markers in the GP2^hi, GP2⁺, GP2^- and E^low populations by global transcriptomic analyses and by RT-qPCR. (C) Expression of NEUROG3 by RT-qPCR in the GP2^hi, 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). DOI: http://dx.doi.org/10.7554/eLife.27564.012

Figure 4—figure supplement 1.. Expression of ductal markers in the GP2^hi, GP2⁺, GP2^- and E^low populations.

Expression of ductal markers in the GP2^hi, GP2⁺, GP2^- and E^low 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). DOI: http://dx.doi.org/10.7554/eLife.27564.013

Figure 5.. Expression of CD142 and SUSD2 in the GP2^hi, GP2⁺, GP2^- and E^low populations.

(A) Expression of CD142 and SUSD2 in GP2⁺, GP2^- and E^low populations at 9WD by microarray analysis. Boxplots were obtained using standardized log2 expression values. (B) Expression of CD142 and SUSD2 in the GP2^hi (in green), GP2⁺ (in red), GP2^- (in purple) and E^low (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 E^low (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 E^lowGP2^-CD142^-SUSD2⁺ (named E^lowSUSD2⁺, in purple) and the E^lowGP2^-CD142^-SUSD2^- (E^lowSUSD2^-, 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. DOI: http://dx.doi.org/10.7554/eLife.27564.014

Figure 5—figure supplement 1.. Expression of CD142 and SUSD2 in the GP2⁺, GP2^- and E^low populations at 8.6WD.

Expression of CD142 and SUSD2 in the GP2⁺ (in red), GP2^- (in purple) and E^low (in orange) populations at 8.6WD by flow cytometry. GP2 and ECAD expressions were gated on live CD45^-CD31^-EPCAM⁺ cells. n = 7. DOI: http://dx.doi.org/10.7554/eLife.27564.015

Figure 6.. Molecular characterization of the GP2^-CD142^-SUSD2^- and E^lowGP2^-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^-), E^lowGP2^-CD142^-SUSD2⁺ (E^lowSUSD2⁺) and E^lowGP2^-CD142^-SUSD2^- (E^lowSUSD2^-) 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^-), E^lowGP2^-CD142^-SUSD2⁺ (E^lowSUSD2⁺) and E^lowGP2^-CD142^-SUSD2^- (E^lowSUSD2^-) subsets. (D, E) Single cell RT-qPCR at 9WD on 172 GP2^-CD142⁺SUSD2^- (GP2^-SUSD2^-, left panel) and 166 E^lowGP2^-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). DOI: http://dx.doi.org/10.7554/eLife.27564.016

Figure 6—figure supplement 1.. Expression of CHGA and NEUROG3 in the E^lowCD142⁺ subsets.

RT-qPCR for CHGA and NEUROG3 in the GP2^-CD142⁺SUSD2^- (named GP2^-CD142⁺), E^lowCD142⁺, E^lowGP2^-CD142^-SUSD2⁺ (named E^lowSUSD2⁺) and M (CD45^-CD45^-EPCAM^-) populations. Heatmaps and RT-qPCR are representative of 3 independents experiments. (mean ± SEM). DOI: http://dx.doi.org/10.7554/eLife.27564.017

Figure 7.. 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 E^low 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-CAD^lowSUSD2⁺ (named E^lowSUSD2⁺), CD142^-E-CAD^lowSUSD2^- (named E^lowSUSD2^-) 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 E^lowSUSD2⁺ and E^lowSUSD2^- 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. DOI: http://dx.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. DOI: http://dx.doi.org/10.7554/eLife.27564.019