Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.

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Annexin A10 is the latest identified member of the annexin family of Ca(2+)- and phospholipid-binding proteins. In previous studies, downregulation of annexin A10 was correlated with dedifferentiation, invasion, and tumor progression, pointing to a possible tumor suppressor role. However, the biochemical characteristics and functions of annexin A10 remain unknown. We show that annexin A10 displays biochemical characteristics atypical for an annexin, indicating a Ca(2+)- and membrane-binding-independent function. Annexin A10 co-localizes with the mRNA-binding proteins SFPQ and PSPC1 at paraspeckles, an only recently discovered nuclear body, and decreases paraspeckle numbers when overexpressed in HeLa cells. In addition, annexin A10 relocates to dark perinucleolar caps upon transcriptional inhibition of RNA polymerase II. We mapped the cap-binding function of annexin A10 to the proximal part of the core domain, which is missing in the short isoform of annexin A10, and show its independence from the remaining functional type II Ca(2+)-binding site. In contrast to this, paraspeckle recruitment required additional core regions and was negatively affected by the mutation of the last type II Ca(2+)-binding site. Additionally, we show that overexpression of annexin A10 in HeLa cells increases their sensitivity to apoptosis and reduces colony formation. The identification of unique nuclear and biochemical characteristics of annexin A10 points towards its membrane-independent role in paraspeckle-associated mRNA regulation or processing.

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Organoid culture is a novel in vitro culture method that promotes the growth of primary and PSC-derived cells in three-dimensional culture to generate structures that recapitulate tissue morphology. We have developed PancreaCult™ Organoid Medium Kits (Human), which combine PancreaCult™ Organoid Initiation Medium (OIM) and serum-free PancreaCult™ Organoid Growth Medium (OGM) into a robust and standardized workflow for the establishment and expansion of pancreatic duct organoids. To establish organoids, fresh or cryopreserved islet-depleted exocrine tissue was seeded in Corning® Matrigel® domes and cultured in PancreaCult™ OIM. After 3 days, the medium was changed to PancreaCult™ OGM and the organoids were passaged and maintained in this medium thereafter, and passaged as fragments every 4 - 7 days. Organoids were successfully established from all 5 donors with a 2- to 6-fold expansion in cell numbers every 7 - 14 days. Real-time PCR and immunocytochemistry demonstrated that these organoids express pancreatic duct markers (PDX1, SOX9, KRT19, CFTR, CA2, and MUC1), as well as proliferation and stem cell markers, Ki-67 and LGR5, respectively. Pancreatic duct organoids could be maintained for at least 10 passages (n = 3) and cryopreserved as fragments that could be used for rapid re-establishment of cultures at later time points. Organoids could also be dissociated and re-seeded in Prostaglandin E2-free PancreaCult™ OGM supplemented with ROCK inhibitor, Y-27632 and 10% FBS on Transwell® inserts to create 2D monolayers. In addition to normal pancreatic duct cultures, PancreaCult™ OGM supported the efficient long-term expansion of 4 pre-established pancreatic ductal adenocarcinoma (PDAC) organoid lines and maintained the mutational profile of the parent line after 5 passages, as determined through whole exome sequencing of 29 cancer-associated genes. Through the optional removal of epidermal growth factor (EGF), PancreaCult™ OGM can be modified to suppress normal cell growth to select for tumor cells with activating KRAS mutations. Our results demonstrate that PancreaCult™ Organoid Medium Kits provide a robust and flexible in vitro culture system for the establishment and expansion of normal pancreatic duct cells, as well as the long-term expansion of PDAC organoids. Citation Format: Nina Quiskamp, Angela Tsai, Joanna Pan, Misha Hasan, George Chang, Isabella T. Tai, Allen C. Eaves, Sharon A. Louis, Ryan K. Conder. PancreaCult™ Organoid Medium Kits support the robust establishment and expansion of human pancreatic duct organoids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A077.