Single-cell profiling of GP2-enriched pancreatic progenitors to simultaneously create acinar, ductal, and endocrine organoids

Abstract

Rationale: Pancreatic lineage specification follows the formation of tripotent pancreatic progenitors (PPs). Current protocols rebuilding PPs in vitro have an endocrine lineage bias and are mostly based on PDX1/NKX6-1 coexpression neglecting other markers decisive for PP heterogeneity and lineage potential. However, true tripotent PPs are of utmost interest to study also exocrine disorders such as pancreatic cancer and to simultaneously generate all three pancreatic lineages from the same ancestor. Methods: Here, we performed a comprehensive compound testing to advance the generation of multipotent progenitors, which were further characterized for their trilineage potential in vitro and in vivo . The heterogeneity and cell-cell communication across the PP subpopulations were analyzed via single-cell transcriptomics. Results: We introduce a novel PP differentiation platform based on a comprehensive compound screening with an advanced design of experiments computing tool to reduce impurities and to increase Glycoprotein-2 expression and subsequent trilineage potential. Superior PP tripotency was proven in vitro by the generation of acinar, endocrine, and ductal cells as well as in vivo upon orthotopic transplantation revealing all three lineages at fetal maturation level. GP2 expression levels at PP stage ascribed varying pancreatic lineage potential. Intermediate and high GP2 levels were superior in generating endocrine and duct-like organoids (PDLO). FACS-based purification of the GP2 high PPs allowed the generation of pancreatic acinar-like organoids (PALO) with proper morphology and expression of digestive enzymes. scRNA-seq confirmed multipotent identity, positioned the GP2/PDX1/NKX6-1 high population next to human fetal tip and trunk progenitors and identified novel ligand-receptor (LR) interactions in distinct PP subpopulations. LR validation experiments licensed midkine and VEGF signaling to increase markers labelling the single cell clusters with high GP2 expression. Conclusion: In this study, we guide human pluripotent stem cells into multipotent pancreatic progenitors. This common precursor population, which has the ability to mature into acinar, ductal and functional β-cells, serves as a basis for studying developmental processes and deciphering early cancer formation in a cell type-specific context. Using single-cell RNA sequencing and subsequent validation studies, we were able to dissect PP heterogeneity and specific cell-cell communication signals.

Glycoprotein 2 as a gut gate keeper for mucosal equilibrium between inflammation and immunity

“…GP2, as a multifunctional protein, is believed to have potential therapeutic applications beyond its role as a diagnostic tool. For instance, as previously mentioned, GP2 serves as a specific marker for pancreatic acinar cells [ 23 , 122 ]. Building upon this foundation, Ameri and colleagues successfully isolated and differentiated functional β cells capable of responding to changes in glucose levels from GP2 + cells [ 123 ].…”

“…GP2, initially discovered in the pancreas by MacDonald and Ronzio, is a member of the zona pellucida (ZP) domain protein family [ 21 ]. Its expression has been observed in certain multipotent pancreatic progenitor cells and pancreatic acinar cells [ 22 , 23 ]. In acinar cells, GP2 is co-located on the glycolipid-enriched ectoleaflet (luminal surface) in apical secretory compartments, including secretory granules [ 24 ].…”

A ONECUT1 regulatory, non-coding region in pancreatic development and diabetes

“…Differentiation of hESCs to pancreatic progenitors was performed as previously reported 33 . In For flow cytometry analysis of NKX6.1 and PDX1 expression on day 13, the protein kinase C activator indolacam V was excluded from the differentiation medium.…”

“…Following the differentiation of hESC into pancreatic progenitors as outlined above, the cells were subsequently guided towards pancreatic endocrine progenitors using a previously established monolayer culture method 33,35 . Briefly, at day 13 of differentiation, medium change was performed with stage 5 monolayer medium supplemented with 1 µM Latrunculin A (Biomol, #Cay10010630).…”

“…Intracellular flow cytometry staining was performed as previously described 33,38 . The following primary antibodies were used: anti-NKX6.1 (1µg/ml, DSHB, F55A12), anti-PDX1…”