Introduction
Genes involved in multiple pancreatic diseases suggest shared disease mechanisms. Recently, we demonstrated that ONECUT1 controls a transcriptional and epigenetic machinery regulating endocrine development involved in a spectrum of diabetes, encompassing monogenic (recessive and dominant) as well as multifactorial inheritance1,2. Interestingly, ONECUT1 mutations causing monogenic recessive syndromic diabetes were not only characterized by endocrine dysfunction but also intrauterine growth retardation, pancreas hypoplasia including exocrine insufficiency and gallbladder agenesis/hypoplasia in line with studies of mouse knockout (KO) models2. The latter rodent models also indicate a ONECUT1 role in maintaining pancreatic epithelial homeostasis and pancreatic cancer progression3. However, detailed knowledge of transcriptional regulation in human pancreas is rather limited.
Aims & Methods
To further unravel the role of ONECUT1 in human pancreatic exocrine development and tissue maintenance, we implemented a human embryonic stem cell (hESC) derived pancreatic duct-like organoid (PDLO) model4. Wildtype (WT) and ONECUT1 KO cell lines were differentiated to pancreatic progenitor cells (PP) and further matured to PDLOs in 3D matrigel culture accompanied by staged ATAC- and RNA-sequencing as well as functional analysis.
Results
Our data indicate that ONECUT1 is not only vital for pancreatic endocrine development, but rather seems to be a key player in establishing all three lineages that contribute to the human pancreas. Loss of the gene leads to altered exocrine development with changed morphology and functionality resulting in big, cyst-like ducts, which was caused by higher proliferation and reduced apoptosis compared to WT PDLOs. Additionally, polarity was impaired in KO PDLOs, resulting in a redistribution of CFTR channels on both the luminal and basal side of the cells. A lineage bias towards ductal cells was observed when PPs were transplanted onto the porcine urinary bladder (PUB)5, resulting in a reduced number of acinar cells and endocrine cells. This is further supported by early expression of maturation marker cytokeratin 7 in PDLO cultures. Moreover, GSEA analysis of RNA-sequencing data highlights a disturbed developmental program in line with results from immunofluorescence staining. Finally, oncogenic activation in KO PDLOs promotes the EMT program supporting its additional role in tissue homeostasis.
Conclusion
In summary, ONECUT1 seems to play a vital role in pancreatic organogenesis and homeostasis. Our in vitro approach directing human ONECUT1-depleted hESCs toward pancreatic ducts recapitulates aspects of patient phenotypes and therefore, provides a valuable methodology to characterize pancreatic diseases.
References
1. Heller, S., et al. Transcriptional changes and the role of ONECUT1 in hPSC pancreatic differentiation. Commun Biol 4, 1298 (2021).
2. Philippi, A., et al. Mutations and variants of ONECUT1 in diabetes. Nature Medicine 27, 1928-1940 (2021).
3. Zhang, H., et al. Multiple, temporal-specific roles for HNF6 in pancreatic endocrine and ductal differentiation. Mechanisms of development 126, 958-973 (2009).
4. Breunig, M., et al. Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells. Cell Stem Cell 28, 1105-1124. e1119 (2021).
5. 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 11, 2102345 (2022).
