Introduction
Human stem cell-based organoids are well established modeling systems widely used to investigate a range of different diseases. For the pancreas, especially endocrine stem cell-derived islets are used to model and decipher the complex development of diabetes. Although we and others also generated pancreatic duct-like organoids (PDLO) [1-4], engineering the main pancreatic compartment, the acinar cells, is underrepresented in literature [1, 4, 5]. Here, we used our advanced differentiation protocol to generate pancreatic progenitors (PPs) [1]. We enriched for the glycoprotein-2 (GP2) expressing multipotent pancreatic progenitors and generated a finetuned acinar differentiation protocol based on pathway and transcriptome analysis of PPs on single cell level. The optimized protocol robustly produces pure mature and functionally active pancreatic acinar-like organoids which we extensively characterized and validated by high resolution imaging, electron microscopy, transplantation studies and functional assays.
Aims & Methods
Immunofluorescence staining of key acinar markers was performed to comprehensively characterize the stem cell-based acinar organoids. High resolution imaging and transmission electron microscopy revealed the presence of secretory vesicles and digestive enzymes. Functionality was proven by enzyme activity assays. Further maturation of the acinar organoids was monitored by transplantation on an ex vivo scaffold, the porcine urinary bladder [6, 7].
Results
In this study we succeeded in producing functional and mature pancreatic acinar organoids by differentiation of human embryonic stem cells. The enrichment for multipotent pancreatic progenitors was facilitated by a double magnetic activated cell sorting, a straightforward approach to sort for GP2 high expressing multipotent progenitors. Finetuning and modulating relevant pathways identified with a PP single-cell RNA sequencing dataset was based on building trajectories, cell-cell communication patterns and analyzing expression profiles. A comprehensive pipeline for characterization and analysis of the resulting acinar organoids validated their functionality and maturity.
Conclusion
The here generated stem-cell derived pancreatic acinar-like organoids provide a useful human model system to investigate pancreatic exocrine disorders, like pancreatic cancer or pancreatitis. It offers a valuable tool for understanding cancer initiation and progression in an animal-reduced human setup.
References
1. Merz, S., et al., Single-cell profiling of GP2-enriched pancreatic progenitors to simultaneously create acinar, ductal, and endocrine organoids. Theranostics, 2023. 13(6): p. 1949.
2. Breunig, M., et al., Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells. Cell Stem Cell, 2021. 28(6): p. 1105-1124. e19.
3. Breunig, M., et al., Differentiation of human pluripotent stem cells into pancreatic duct-like organoids.STAR protocols, 2021. 2(4): p. 100913.
4. Huang, L., et al., Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids. Cell Stem Cell, 2021. 28(6): p. 1090-1104. e6.
5. Hohwieler, M., et al., Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling. Gut, 2017. 66(3): p. 473-486.
6. Melzer, M.K., et al., Organoids at the PUB: the porcine urinary bladder serves as a pancreatic niche for advanced cancer modeling. Advanced Healthcare Materials, 2022. 11(11): p. 2102345.
7. Melzer, M.K., et al., Protocol to use de-epithelialized porcine urinary bladder as a tissue scaffold for propagation of pancreatic cells. STAR protocols, 2022. 3(4): p. 101869.
