Mechanistic elucidation of human pancreatic acinar development using single-cell transcriptome analysis on a human iPSC differentiation model

Abstract

Few effective treatments have been developed for intractable pancreatic exocrine disorders due to the lack of suitable disease models using human cells. Pancreatic acinar cells differentiated from human induced pluripotent stem cells (hiPSCs) have the potential to solve this issue. In this study, we aimed to elucidate the developmental mechanisms of pancreatic exocrine acinar lineages to establish a directed differentiation method for pancreatic acinar cells from hiPSCs. hiPSC-derived pancreatic endoderm cells were spontaneously differentiated into both pancreatic exocrine and endocrine tissues by implantation into the renal subcapsular space of NOD/SCID mice. Single-cell RNA-seq analysis of the retrieved grafts confirmed the differentiation of pancreatic acinar lineage cells and identified REG4 as a candidate marker for pancreatic acinar progenitor cells. Furthermore, differential gene expression analysis revealed upregulated pathways, including cAMP-related signals, involved in the differentiation of hiPSC-derived pancreatic acinar lineage cells in vivo, and we found that a cAMP activator, forskolin, facilitates the differentiation from hiPSC-derived pancreatic endoderm into pancreatic acinar progenitor cells in our in vitro differentiation culture. Therefore, this platform contributes to our understanding of the developmental mechanisms of pancreatic acinar lineage cells and the establishment of differentiation methods for acinar cells from hiPSCs.

Keywords: Acinar cell; Forskolin; Pancreas; REG4; Single-cell transcriptome; iPSC.

Fig. 1

In vivo differentiation of hiPSC-derived pancreatic endoderm cells mimics early pancreatic development. (A) A schematic representation of induction of hiPSC-derived pancreatic endoderm cells and implantation into immunodeficient mice. A, activin A; C, CHIR99021; Y, Y-27632; K, KGF; N, Noggin; KC, KAAD-cyclopamine; TT, TTNPB; E, EGF. (B) Immunostaining of hiPSC-derived cells on Stage 4 Day 6 (total day 17) before implantation for PDX1 (green), NKX6.1 (red), and nuclei (blue). (C) Induction efficiency of hiPSC-derived PDX1⁺NKX6.1⁺ pancreatic endoderm cells on Stage 4 Day 6 (total day 17) before implantation, as evaluated by flow cytometry. Data from three independent experiments are presented as mean ± SD (n = 3). (D) Macroscopic images of grafts under the left renal capsule of a NOD/SCID mouse sacrificed 30 days after implantation (upper panel) and following isolation from the host mouse kidney (lower panel). Blue arrows indicate grafts. (E) Immunostaining of grafts on the indicated days after implantation for CPA1 (green) and NKX6.1 (red; upper panels), Mucin 1 (green) and E-CADHERIN (red; middle panels), and PRSS1 (green) and nuclei (blue; lower panels). Note that background signals were found in the host mouse kidney (MK). Scale bars: 100 μm in (B) and (E) and 5 mm in (D).

Fig. 2

Transition dynamics of differentiation states from pancreatic endoderm cells to pancreatic acinar cells revealed by scRNA-seq. (A) Clustering of cells using the tSNE method (left panel) and distribution of cells on the indicated days (right panels). Only the six clusters annotated as pancreatic cells (clusters 0, 1, 2, 8, 10, and 12) in Fig. S4 (n = 96 for pancreatic endoderm aggregates, n = 8 for day 11 grafts, and n = 5 for day 25 grafts) were reclustered. (B) Distribution of cells expressing marker genes for pancreatic endoderm cells (PDX1, NKX6.1, PTF1A, and SOX9), pancreatic endocrine cells (CHGA, INS, and GCG), and pancreatic acinar cells (PRSS1, SPINK1, CPA1, CPA2, CPB1, and CTRC). (C) The direction and magnitude of differentiation stage transition among cell populations by RNA velocity. The lower boxed panel shows the velocity map by selecting clusters 5, 11, and 12. (D) Visualizing the differentiation stage transition from pancreatic endoderm to acinar lineage cells (upper panel) and changes in the expression of pancreatic acinar marker genes during the differentiation process (lower panel) by pseudotime analysis.

Fig. 3

Identification of novel marker candidates for pancreatic acinar progenitor cells. (A) Dot plots of DEGs (|log2FC|> 1, P value < 0.01) among clusters of pancreatic endoderm cells (cluster 12), acinar progenitor cells (cluster 11), acinar cells (cluster 5), endocrine cells (cluster 2), and duct cells (cluster 3). (B) Volcano plots of DEGs (|log2FC|> 1, P value < 0.01) comparing gene expression of pancreatic endoderm cells (cluster 12) versus acinar progenitor cells (cluster 11; left panel) and acinar progenitor cells (cluster 11) versus acinar cells (cluster 5; middle panel) and the Venn diagram (right panel). (C) Distribution of cells expressing the four candidate markers for acinar progenitor cells, REG4, BGN, COL1A2, and HAPLN1. (D) Expression pattern of REG4 and PRSS1 among the 3 clusters based on DEGs (|log2FC|> 1, P value < 0.01).

Fig. 4

Induction from hiPSC-derived pancreatic endoderm to pancreatic acinar progenitor cells. (A) Candidate factors (left) and signaling pathways (right) involved in the differentiation of pancreatic acinar progenitor cells, as identified using pathway analysis by IPA. (B) Comparison of effects of 9 candidate differentiation-inducing factors on the expression of REG4 and PRSS1, as evaluated by qRT-PCR. The treatment started on Stage 4 Day 6 (total day 17) and continued for 8 days, ending on total day 25. The following three concentrations were examined for each factor: 10, 50, and 100 ng/mL EGF; 0.2, 1, and 2 ng/mL IL-1B; 10, 50, and 100 ng/mL HGF; 10, 50, and 100 µM pCPT-cAMP (pC); 2, 10, and 20 nM orskolin (FS); 10, 50, and 100 ng/mL FGF2; 0.02, 0.1, and 0.2 nM bexarotene (B); 5, 25, and 50 ng/mL IGF1; and 1, 5, and 10 µg/mL lipopolysaccharide (LPS). PE, pancreatic endoderm. Red arrows indicate factors that upregulated the expression of REG4 or PRSS1 more than 1.5 times compared to the untreated control (Control). Data from three independent experiments are presented as mean ± SD (n = 3). We normalized the expression values against the house keeping gene GAPDH and then against the untreated control. (C) Effects of treatment with 20 nM forskolin on the expression of acinar lineage markers, REG4, PTF1A, CPA1, PRSS1, SPINK1, and CTRC, as evaluated by qRT-PCR. The treatment started on Stage 4 Day 6 (total day 17) and continued for 8 days, ending on total day 25. Graft indicates one graft sample on day 30 after implantation, which differs from grafts used for scRNA-seq. The graft sample on day 30 after implantation was isolated from the host renal parenchyma and capsule, and cells were dispersed in a collagenase/dispase solution for 15 min at 37 °C, dissociated into single cells by gentle pipetting, and used for RNA isolation without sorting for human cells. Data from three independent experiments are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 by two-tailed Student’s t-test. We normalized the expression values against GAPDH and then against the untreated control (Control). (D) Immunostaining of day 30 grafts from hiPSC-derived pancreatic endoderm cells with or without forskolin treatment for PRSS1 (green) and nuclei (blue; left and upper right panels), and CPA1 (green) and NKX6.1 (red; middle right panels) and quantification of the percentage (%) of PRSS1⁺ or CPA1⁺ cells from all cells within the engraftment region (lower right panel). Data from three independent experiments are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 by two-tailed Student’s t-test. Scale bars: 300 μm in (D; left panels) and 100 μm in (D; right panels).