Single-cell transcriptome analysis of NEUROG3+ cells during pancreatic endocrine differentiation with small molecules

Area of Science:

• Stem cell biology and regenerative medicine.
• Endocrinology and diabetes research.
• Single-cell genomics and transcriptomics.

Background:

• Efficient differentiation of human embryonic stem cells (hESCs) into NEUROG3+ pancreatic endocrine cells is crucial for diabetes stem cell therapy.
• Current methods face bottlenecks in achieving high efficiency and specific cell fate decisions.
• Understanding cell properties and fate during differentiation is essential for optimizing protocols.

Discussion:

• The DAPT + 4FS (nicotinamide, dexamethasone, forskolin, Alk5 inhibitor II) method significantly increased NEUROG3 expression to 34.3% in differentiating hESCs.
• Increased NEUROG3+ cells were predominantly found in Insulin+Glucagon+ (INS+GCG+) and SLC18A1+Chromogranin A+ (SLC18A1+CHGA+) populations.
• This suggests enhanced differentiation towards pancreatic endocrine and enterochromaffin-like cells.

Key Insights:

• Single-cell transcriptome analysis revealed that elevated NEUROG3 expression promotes pancreatic endocrine and enterochromaffin-like cell differentiation.
• Gene set enrichment analysis (GSEA) identified potential signaling pathways for optimization: up-regulate G protein-coupled receptor (GPCR) and mitogen-activated protein kinase (MAPK) signals.
• Down-regulating Wnt, NIK/NF-KappaB, and cytokine-mediated pathways may further enhance differentiation. PLCE1 can be targeted to modulate GPCR signaling for increased NEUROG3+ cells in INS+GCG+ populations.

Outlook:

• ALCAMhigh CD9low can serve as a marker for identifying endocrine cell populations.
• ALCAMhigh CD9lowCDH1low markers can be used to exclude non-pancreatic endocrine SLC18A1+CHGA+ cells.
• These findings provide a foundation for refining hESC differentiation protocols for diabetes therapy and advancing pancreatic islet cell generation.