Engineering human ducts and acini from pluripotent stem cells to study cell type of origin and tumour evolution in pancreatic cancer
Final Report Abstract
Our project aimed at developing protocols for exclusive pancreatic exocrine lineage commitment, focusing on acinar and ductal lineages. The project was structured around several investigational pillars. First, we optimized protocols to differentiate human pluripotent stem cells (hPSCs) into multipotent pancreatic progenitors (PPs) with minimal nonpancreatic contamination. The resulting GP2-positive PPs showed enhanced endocrine differentiation, producing functional β-like cells. FACS-purified GP2high PPs demonstrated true trilineage potential, maturing into acinar, ductal, and endocrine organoids. Second, we developed a two-phase screening tool to differentiate hPSCs into PDLOs, providing a genetic model for dysplasia and early pancreatic cancer. Also, a protocol for pancreatic acinar-like organoids has been generated. Our experiments with oncogenic KRAS and CDKN2A loss in PDLOs revealed early tumor development changes setting stage for the first-in-kind dysplasia model. Third, we engineered a microwell chip for creating 3D aggregates of hPSC-derived PPs, enabling precise differentiation into PDLOs, high-resolution imaging, single-cell RNA sequencing, and secretome analysis. This chip identified potential prognostic markers for pancreatic ductal adenocarcinoma. Forth, we used the porcine urinary bladder (PUB) model - awarded by the DFG - to replicate pancreatic ductal structures with human cells, culture patient-derived organoids, and conduct cost-effective drug testing. The PUB model proved to be a versatile platform for studying tumor evolution and screening drugs. Finally, the PDLO system was licensed to dissect developmental features of ductal lineage entry and to test pancreatic cancer tumor markers. In sum, our project successfully developed protocols and technologies allowing mutually exclusive pancreatic lineage segregation, created models for early pancreatic dysplasia, and optimized cancer research methodologies, potentially contributing significantly to early diagnoses as well as the understanding and treatment of pancreatic diseases.
Publications
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CDKN2A-Mutated Pancreatic Ductal Organoids from Induced Pluripotent Stem Cells to Model a Cancer Predisposition Syndrome. Cancers, 13(20), 5139.
Merkle, Jessica; Breunig, Markus; Schmid, Maximilian; Allgöwer, Chantal; Krüger, Jana; Melzer, Michael K.; Bens, Susanne; Siebert, Reiner; Perkhofer, Lukas; Azoitei, Ninel; Seufferlein, Thomas; Heller, Sandra; Meier, Matthias; Müller, Martin; Kleger, Alexander & Hohwieler, Meike
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Differentiation of human pluripotent stem cells into pancreatic duct-like organoids. STAR Protocols, 2(4), 100913.
Breunig, Markus; Merkle, Jessica; Melzer, Michael Karl; Heller, Sandra; Seufferlein, Thomas; Meier, Matthias; Hohwieler, Meike & Kleger, Alexander
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Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells. Cell Stem Cell, 28(6), 1105-1124.e19.
Breunig, Markus; Merkle, Jessica; Wagner, Martin; Melzer, Michael K.; Barth, Thomas F.E.; Engleitner, Thomas; Krumm, Johannes; Wiedenmann, Sandra; Cohrs, Christian M.; Perkhofer, Lukas; Jain, Gaurav; Krüger, Jana; Hermann, Patrick C.; Schmid, Maximilian; Madácsy, Tamara; Varga, Árpád; Griger, Joscha; Azoitei, Ninel; Müller, Martin ... & Kleger, Alexander
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Single-cell-resolved differentiation of human induced pluripotent stem cells into pancreatic duct-like organoids on a microwell chip. Nature Biomedical Engineering, 5(8), 897-913.
Wiedenmann, Sandra; Breunig, Markus; Merkle, Jessica; von Toerne, Christine; Georgiev, Tihomir; Moussus, Michel; Schulte, Lucas; Seufferlein, Thomas; Sterr, Michael; Lickert, Heiko; Weissinger, Stephanie Ellen; Möller, Peter; Hauck, Stefanie M.; Hohwieler, Meike; Kleger, Alexander & Meier, Matthias
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Organoids at the PUB: The Porcine Urinary Bladder Serves as a Pancreatic Niche for Advanced Cancer Modeling. Advanced Healthcare Materials, 11(11).
Melzer, Michael Karl; Breunig, Markus; Arnold, Frank; Wezel, Felix; Azoitei, Anca; Roger, Elodie; Krüger, Jana; Merkle, Jessica; Schütte, Lena; Resheq, Yazid; Hänle, Mark; Zehe, Viktor; Zengerling, Friedemann; Azoitei, Ninel; Klein, Lukas; Penz, Frederike; Singh, Shiv K.; Seufferlein, Thomas; Hohwieler, Meike ... & Kleger, Alexander
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DNA methylation‐associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis. The Journal of Pathology, 261(2), 139-155.
Krüger, Jana; Fischer, Anja; Breunig, Markus; Allgöwer, Chantal; Schulte, Lucas; Merkle, Jessica; Mulaw, Medhanie A.; Okeke, Nnamdi; Melzer, Michael K.; Morgenstern, Clara; Azoitei, Ninel; Seufferlein, Thomas; Barth, Thomas FE; Siebert, Reiner; Hohwieler, Meike & Kleger, Alexander
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Single-cell profiling of GP2-enriched pancreatic progenitors to simultaneously create acinar, ductal, and endocrine organoids. Theranostics, 13(6), 1949-1973.
Merz, Sarah; Breunig, Markus; Melzer, Michael Karl; Heller, Sandra; Wiedenmann, Sandra; Seufferlein, Thomas; Meier, Matthias; Krüger, Jana; Mulaw, Medhanie A.; Hohwieler, Meike & Kleger, Alexander
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TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration. BMC Biology, 21(1).
Melzer, Michael Karl; Schirge, Silvia; Gout, Johann; Arnold, Frank; Srinivasan, Dharini; Burtscher, Ingo; Allgöwer, Chantal; Mulaw, Medhanie; Zengerling, Friedemann; Günes, Cagatay; Lickert, Heiko; Christoffels, Vincent M.; Liebau, Stefan; Wagner, Martin; Seufferlein, Thomas; Bolenz, Christian; Moon, Anne M.; Perkhofer, Lukas & Kleger, Alexander
