Generation of vascularized pancreatic progenitors through co-differentiation of endoderm and mesoderm from human pluripotent stem cells

Abstract

Background: The simultaneous differentiation of human pluripotent stem cells (hPSCs) into both endodermal and mesodermal lineages is crucial for developing complex, vascularized tissues, yet poses significant challenges. This study explores a method for co-differentiation of mesoderm and endoderm, and their subsequent differentiation into pancreatic progenitors (PP) with endothelial cells (EC).

Methods: Two hPSC lines were utilized. By manipulating WNT signaling, we optimized co-differentiation protocols of mesoderm and endoderm through adjusting the concentrations of CHIR99021 and mTeSR1. Subsequently, mesoderm and endoderm were differentiated into vascularized pancreatic progenitors (vPP) by adding VEGFA. The differentiation characteristics and potential of vPPs were analyzed via transcriptome sequencing and functional assays.

Results: A low-dose CHIR99021 in combination with mTeSR1 yielded approximately 30% mesodermal and 70% endodermal cells. Introduction of VEGFA significantly enhanced EC differentiation without compromising PP formation, increasing the EC proportion to 13.9%. Transcriptomic analyses confirmed the effectiveness of our protocol, showing up-regulation of mesodermal and endothelial markers, alongside enhanced metabolic pathways. Functional assays demonstrated that vPPs could efficiently differentiate into insulin-producing β-cells, as evidenced by increased expression of β-cell markers and insulin secretion.

Conclusion: Our findings provide a robust method for generating vPPs, which holds significant promise for regenerative medicine applications, particularly in diabetes treatment.

Keywords: Endoderm differentiation; Human pluripotent stem cells; Mesoderm differentiation; Multi-lineage co-differentiation; Vascularized pancreatic progenitors.

Fig. 1

Optimization of co-differentiation of endoderm and mesoderm from hPSC. (A) A schematic representation of the differentiation protocol from Day 0 to Day 3. The endoderm differentiation scheme was adapted from Chen et al. Our optimized protocol facilitates the simultaneous induction of both endoderm and mesoderm. (Created in BioRender: https://BioRender.com/j57p699 ). (B) Immunofluorescence and flow cytometry analysis confirmed that most Day 0 hPSCs expressed NANOG and OCT4. Scale bar, 50 μm. (C) Quantitative PCR analysis. High levels of CHIR99021 promote the optimal proportion of mesodermal and endodermal co-differentiation compared to protocols with increased or decreased CHIR99021 concentrations. (D) Quantitative PCR analysis, (E) flow cytometry, and (F) immunofluorescence showed that 2.5% mTeSR1 induces an appropriate proportion of mesoderm and endoderm. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns: no statistical differences)

Fig. 2

Generation of Vascularized Pancreatic Progenitors (vPP). (A) A schematic representation of the PP and vPP differentiation protocol. The PP differentiation scheme was adapted from Chen et al. Our vPP protocol facilitates the simultaneous induction of both EC and PP from Day 4 to Day 10. (Created in BioRender: https://BioRender.com/d21p796). (B) Flow cytometry analysis of EC marker CD34. (C and D) Quantitative PCR analysis of EC markers (CD34, CD31, CDH5), PP markers (PDX1, SOX9, NKX6.1), and pericyte markers (PDGFRβ, α-SMA). (E) Immunostaining of generated PP (day 10) against PDX1 and CD31, comparing the vPP and Chen’s protocols. Scale bar, 250 μm. (F) Cell counting analysis. Statistical analysis of cell counts after re-inoculation for 3 days with an equal number of cells on Day 10 under both protocols. (G) Immunostaining of generated PP (day 10) against Ki67, comparing the vPP and Chen’s protocol. Scale bar, 100 μm. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns: no statistical differences)

Fig. 3

Characterization of endoderm and mesoderm co-differentiation via RNA sequencing. (A) Heatmap representation of DEGs of endoderm and mesoderm co-differentiation from Optimized Protocol compared to control protocol (Chen’s Protocol). (B) Gene Ontology (GO) enrichment analysis of DEGs involved in mesoderm and endoderm development. (C and D) Up-regulation of mesoderm-associated genes and down-regulation of endoderm markers in the optimized protocol. (E and F) Gene set enrichment analysis (GSEA) indicating significant up-regulation of mesoderm formation and down-regulation of endodermal cell differentiation. (G) GSEA for KEGG pathway analysis

Fig. 4

Characterization of vascularized pancreatic progenitors (vPP) via RNA sequencing. (A) Heatmap showing upregulated endothelium development-related genes in vPP from vPP Protocol compared to PP from Chen’s protocol. (B) GO enrichment analysis of up-regulated DEGs in vPP. (C) Up-regulation and down-regulation of endothelium development-related genes in vPP protocol, compared to Chen’s protocol. (D) GSEA results suggesting vPP protocol promotes processes of hematopoietic cell lineage, vasculature development, and angiogenesis

Fig. 5

Transcription factor analysis for vascularized pancreatic progenitors (vPP). (A) Volcano plot for differentially expressed TF genes of endoderm and mesoderm co-differentiation from Optimized Protocol compared to control protocol (Chen’s Protocol) on day 3. (B) Volcano plot for differentially expressed TF genes in vPP Protocol compared to PP from Chen’s protocol on day 10. (C) GO enrichment and gene correlation analysis of up-regulated differentially expressed TF genes associated with mesoderm development and vasculogenesis on day 3. (D) GO enrichment and gene correlation analysis of up-regulated differentially expressed TF genes associated with mesoderm development and vasculogenesis on day 10

Fig. 6

Differentiation of vPP into β-Cells. (A) Quantitative PCR analysis of mature β-cells (NKX6.1, INS and MAFB), immature β-cells (PDX1 and SOX9), and ECs (CD34) at day 27. (B) Immunofluorescence images showing co-expression of INS and MAFA in vPP-derived β-cells at day 27. (C) Flow cytometry analysis of insulin-producing β-cells (C-peptide⁺ cells), indicating a higher ratio in vPP-derived β-cells compared to the control. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns: no statistical differences)