DNA methylation-associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis.

Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct-like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site-specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct-like organoids. Allelic reactivation was also detected in single-cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression-free survival in gemcitabine/nabPaclitaxel-treated and opposing trends in FOLFIRINOX-treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Values and Perspectives of Organoids in the Field of Metabolic Syndrome.

Metabolic syndrome (MetS) has become a global health problem, and the prevalence of obesity at all stages of life makes MetS research increasingly important and urgent. However, as a comprehensive and complex disease, MetS has lacked more appropriate research models. The advent of organoids provides an opportunity to address this issue. However, it should be noted that organoids are still in their infancy. The main drawbacks are a lack of maturity, complexity, and the inability to standardize large-scale production. Could organoids therefore be a better choice for studying MetS than other models? How can these limitations be overcome? Here, we summarize the available data to present current progress on pancreatic and hepatobiliary organoids and to answer these open questions. Organoids are of human origin and contain a variety of human cell types necessary to mimic the disease characteristics of MetS in their development. Taken together with the discovery of hepatobiliary progenitors in situ, the dedifferentiation of beta cells in diabetes, and studies on hepatic macrophages, we suggest that promoting endogenous regeneration has the potential to prevent the development of end-stage liver and pancreatic lesions caused by MetS and outline the direction of future research in this field.

Generation of Human Stem Cell-Derived Pancreatic Organoids (POs) for Regenerative Medicine.

Insulin-dependent diabetes mellitus or type 1 diabetes mellitus (T1DM) is an auto-immune condition characterized by the loss of pancreatic ß-cells. The curative approach for highly selected patients is the pancreas or the pancreatic islet transplantation. Nevertheless, these options are limited by a growing shortage of donor organs and by the requirement of immunosuppression.Xenotransplantation of porcine islets has been extensively investigated. Nevertheless, the strong xenoimmunity and the risk of transmission of porcine endogenous retroviruses, have limited their application in clinic. Generation of ß-like cells from stem cells is one of the most promising strategies in regenerative medicine. Embryonic, and more recently, adult stem cells are currently the most promising cell sources exploited to generate functional ß-cells in vitro. A number of studies demonstrated that stem cells could generate functional pancreatic organoids (POs), able to restore normoglycemia when implanted in different preclinical diabetic models. Nevertheless, a gradual loss of function and cell dead are commonly detected when POs are transplanted in immunocompetent animals. So far, the main issue to be solved is the post-transplanted islet loss, due to the host immune attack. To avoid this hurdle, nanotechnology has provided a number of polymers currently under investigation for islet micro and macro-encapsulation. These new approaches, besides conferring PO immune protection, are able to supply oxygen and nutrients and to preserve PO morphology and long-term viability.Herein, we summarize the current knowledge on bioengineered POs and the stem cell differentiation platforms. We also discuss the in vitro strategies used to generate functional POs, and the protocols currently used to confer immune-protection against the host immune attack (micro- and macro-encapsulation). In addition, the most relevant ongoing clinical trials, and the most relevant hurdles met to move towards clinical application are revised.

Emerging Diabetes Therapies: Regenerating Pancreatic ß Cells.

The incidence of diabetes mellitus (DM) is steadily increasing annually, with 537 million diabetic patients as of 2021. Restoring diminished ß cell mass or impaired islet function is crucial in treating DM, particularly type 1 DM. However, the regenerative capacity of islet ß cells, which primarily produce insulin, is severely limited, and natural regeneration is only observed in young rodents or children. Hence, there is an urgent need to develop advanced therapeutic approaches that can regenerate endogenous ß cells or replace them with stem cell (SC)-derived or engineered ß-like cells. Current strategies for treating insulin-dependent DM mainly include promoting the self-replication of endogenous ß cells, inducing SC differentiation, reprogramming non-ß cells into ß-like cells, and generating pancreatic-like organoids through cell-based intervention. In this Review, we discuss the current state of the art in these approaches, describe associated challenges, propose potential solutions, and highlight ongoing efforts to optimize ß cell or islet transplantation and related clinical trials. These effective cell-based therapies will generate a sustainable source of functional ß cells for transplantation and lay strong foundations for future curative treatments for DM.

Application and challenge of pancreatic organoids in therapeutic research.

The in-vivo non-human primate animal and in-vitro cell disease models play a crucial part in the study of the mechanisms underlying the occurrence and development of pancreatic diseases, but with increasingly prominent limitations with in-depth research. Organoids derived from human pluripotent and adult stem cells resemble human in-vivo organs in their cellular composition, spatial tissue structure and physiological function, making them as an advantageous research tool. Up until now, numerous human organoids, including pancreas, have been effectively developed, demonstrating significant potential for research in organ development, disease modeling, drug screening, and regenerative medicine. However, different from intestine, liver and other organs, the pancreas is the only special organ in the human body, consisting of an exocrine gland and an endocrine gland. Thus, the development of pancreatic organoid technology faces greater challenges, and how to construct a composite pancreatic organoid with exocrine and endocrine gland is still difficult in current research. By reviewing the fundamental architecture and physiological role of the human pancreas, along with the swiftly developing domain of pancreatic organoids, we summarize the method and characteristics of human pancreatic organoids, and its application in modeling pancreatic diseases, as a platform for individualized drug screening and in regenerative medicine study. As the first comprehensive review that focus on the pharmacological study of human pancreatic organoid, the review hopes to help scholars to have a deeper understanding in the study of pancreatic organoid.

Pancreatic Cancer Organoids: An Emerging Platform for Precision Medicine?

Despite recent therapeutic advances, pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies, with remarkable resistance to treatment, poor prognosis, and poor clinical outcome. More efficient therapeutic approaches are urgently needed to improve patients' survival. Recently, the development of organoid culture systems has gained substantial attention as an emerging preclinical research model. PDAC organoids have been developed to study pancreatic cancer biology, progression, and treatment response, filling the translational gap between in vitro and in vivo models. Here, we review the rapidly evolving field of PDAC organoids and their potential as powerful preclinical tools that could pave the way towards precision medicine for pancreatic cancer.

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

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 GP2high 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/NKX6-1high 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 ß-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.

A mouse pancreatic organoid model to compare PD-L1 blocking antibodies.

Immune checkpoint inhibitors (ICIs) have changed the therapeutic landscape for cancer patients, but diabetes, a rare, severe immune-related endocrinopathy, is linked to ICI therapy. It is unclear whether glycosylation of ICIs may play a role in the development of this adverse event and how the physiological effects of different ICIs on pancreatic cells should be evaluated. We used a mouse pancreatic organoid model to compare three PD-L1 blocking antibodies in the presence or absence of IFNγ using a metabolic bioanalyzer. Modulation of ICI glycosylation altered its metabolic effects on mouse pancreatic organoids, suggesting that this model could be used to monitor and compare ICIs and to study the mechanisms underlying the development of IC-mediated diabetes.

CDKN2A-Mutated Pancreatic Ductal Organoids from Induced Pluripotent Stem Cells to Model a Cancer Predisposition Syndrome.

Patient-derived induced pluripotent stem cells (iPSCs) provide a unique platform to study hereditary disorders and predisposition syndromes by resembling germline mutations of affected individuals and by their potential to differentiate into nearly every cell type of the human body. We employed plucked human hair from two siblings with a family history of cancer carrying a pathogenic CDKN2A variant, P16-p.G101W/P14-p.R115L, to generate patient-specific iPSCs in a cancer-prone ancestry for downstream analytics. The differentiation capacity to pancreatic progenitors and to pancreatic duct-like organoids (PDLOs) according to a recently developed protocol remained unaffected. Upon inducible expression of KRASG12Dusing a piggyBac transposon system in CDKN2A-mutated PDLOs, we revealed structural and molecular changes in vitro, including disturbed polarity and epithelial-to-mesenchymal (EMT) transition. CDKN2A-mutated KRASG12DPDLO xenotransplants formed either a high-grade precancer lesion or a partially dedifferentiated PDAC-like tumor. Intriguingly, P14/P53/P21 and P16/RB cell-cycle checkpoint controls have been only partly overcome in these grafts, thereby still restricting the tumorous growth. Hereby, we provide a model for hereditary human pancreatic cancer that enables dissection of tumor initiation and early development starting from patient-specific CDKN2A-mutated pluripotent stem cells.

Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells.

Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.

The science and engineering of stem cell-derived organoids-examples from hepatic, biliary, and pancreatic tissues.

The field of organoid engineering promises to revolutionize medicine with wide-ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modelling, cellular therapy, and human development. Organoids are a three-dimensional (3D) miniature representation of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools with which to model organ function. The biological basis for organoids is that they foster stem cell self-renewal, differentiation, and self-organization, recapitulating 3D tissue structure or function better than two-dimensional (2D) systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide a context and rationale for organoids of the liver, pancreas, and gall bladder. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide a historical context, and review a wide range of both biological and mathematical perspectives that enhance understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. We discuss non-invasive assessment of organoids, which can reveal the underlying biological mechanisms and enable improved assays for growth, metabolism, and function. Applications of organoids in cell therapy are also discussed. Taken together, we establish a broad scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids.

Multi-site Neurogenin3 Phosphorylation Controls Pancreatic Endocrine Differentiation.

The proneural transcription factor Neurogenin3 (Ngn3) plays a critical role in pancreatic endocrine cell differentiation, although regulation of Ngn3 protein is largely unexplored. Here we demonstrate that Ngn3 protein undergoes cyclin-dependent kinase (Cdk)-mediated phosphorylation on multiple serine-proline sites. Replacing wild-type protein with a phosphomutant form of Ngn3 increases α cell generation, the earliest endocrine cell type to be formed in the developing pancreas. Moreover, un(der)phosphorylated Ngn3 maintains insulin expression in adult ß cells in the presence of elevated c-Myc and enhances endocrine specification during ductal reprogramming. Mechanistically, preventing multi-site phosphorylation enhances both Ngn3 stability and DNA binding, promoting the increased expression of target genes that drive differentiation. Therefore, multi-site phosphorylation of Ngn3 controls its ability to promote pancreatic endocrine differentiation and to maintain ß cell function in the presence of pro-proliferation cues and could be manipulated to promote and maintain endocrine differentiation in vitro and in vivo.