Introduction

Gene regulatory networks play a vital role in pancreatic development and function. However, pathomechanisms associated with the non-coding genome are just starting to be discovered. Our recent work demonstrated that mutations in the ONECUT1 gene are responsible for a new neonatal diabetes syndrome in homozygous patients, whereas heterozygous carriers develop early-onset diabetes. Stage-specific characterization of in vitro differentiated human embryonic stem cells (hESC) revealed that ONECUT1 plays an important role in transcriptional regulation during pancreatic progenitor formation and the initiation of the endocrine developmental program. Nonetheless, the complex regulation is only partly uncovered. While investigating the ONECUT1 locus, we found a variant most strongly associated with T2D (rs2440374). This variant is located within the lncRNA RP11-209K10.2 (further lncRNA-RP11), 11 kbp distant to ONECUT1. GTEx Portal database shows decreased lncRNA-RP11 expression in the pancreas in the presence of the T2D-associated allele. Also, this SNP disrupts a binding motif for transcription factor NKX2.2 essential for endocrine pancreas development and pancreatic islet-cell fate. Since promoters of lncRNAs can also act as enhancer elements stimulating the regulation of neighboring genes, we hypothesize that the lncRNA-RP11 locus plays an important role in regulating ONECUT1 and that disturbance of this transcriptional network promotes diabetes onset.

Aims & Methods

After deletion of lncRNA-RP11, hESC were differentiated into the pancreatic endocrine lineage followed by characterization of the developmental stages.

Results

Although differentiation of pancreatic progenitors is not impaired by the loss of the regulatory region, stem cell-derived islets further generated from these progenitors exhibit reduced number and function of beta-cells supporting an involvement during pancreas differentiation. The transcript of lncRNA-RP11 with main localization in the nucleus follows similar stage-specific dynamics as ONECUT1 allowing for several modes of action.

Conclusion

Next, a comprehensive mechanistic characterization will provide insights into the exact function of lncRNA-RP11 in pancreatic endocrine development and pathophysiology. Thus, identifying novel molecular mechanisms involved in diabetes will improve our understanding of the transcriptional landscape and lay the groundwork for personalized diabetes treatment.

References

Philippi, A., Heller, S., Costa, I.G. et al. Mutations and variants of ONECUT1 in diabetes. Nat Med 27, 1928–1940 (2021). https://doi.org/10.1038/s41591-021-01502-7

Introduction

Rare early onset cases of diabetes stem from single gene mutations, whereas frequent gene variants can also contribute to multifactorial diabetes, indicating shared disease mechanisms. Our recent study has identified ONECUT1 as a gene implicated in both monogenic and multifactorial diabetes, establishing it as crucial target for investigating fundamental signaling pathways in diabetes pathology [1,2]. However, numerous diabetic characteristics remain unexplained by typical genetic alterations, highlighting the underestimated significance of the non-coding genome. We have identified a neonatal diabetes patient carrying a heterozygous ONECUT1 truncating variant. Remarkably, the patient exhibited a phenotype akin to individuals with homozygous ONECUT1 truncating mutations. Upon further examination, we detected an additional heterozygous deletion in the non-coding genome, leading us to hypothesize that it could impact the regulation of the remaining ONECUT1 allele. Consequently, the dysregulation of the ONECUT1-dependent transcriptional machinery crucial for proper beta-cell development occurs, ultimately leading to the severe diabetic phenotype observed in the patient.

Aims & Methods

Following deletion of specific genomic regions in human embryonic stem cells, we initiated their differentiation into the pancreatic endocrine lineage and meticulously characterized the developmental stages.

Results

Importantly, we successfully replicated the patient’s diabetic phenotype, elucidating deficiencies in both pancreatic development and beta-cell functionality. The differentiation of pancreatic progenitors was notably hindered by the loss of the regulatory region associated with nearly complete ablation of ONECUT1. Moreover, the stem cell-derived islets exhibited a reduced number of beta-cells as well as impaired insulin secretion, even in heterozygous knockouts.

Conclusion

Altogether, our ongoing comprehensive mechanistic characterization provides insights into the precise role of the identified regulatory element in pancreatic beta-cell development and diabetes pathophysiology, emphasizing the significance of the non-coding genome. By unveiling unknown molecular pathomechanisms linked to alterations in regulatory sequences, we contribute to a deeper understanding of the pancreatic transcriptional network and establish the foundation for personalized diabetes treatment.

References

[1] Philippi, A., Heller, S., Costa, I.G. et al. Mutations and variants of ONECUT1 in diabetes. Nat Med 27, 1928–1940 (2021). https://doi.org/10.1038/s41591-021-01502-7
[2] Heller, S., Li, Z., Lin, Q. et al. Transcriptional changes and the role of ONECUT1 in hPSC pancreatic differentiation. Commun Biol 4, 1298 (2021). https://doi.org/10.1038/s42003-021-02818-3

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.

Introduction

Long non-coding RNAs (lncRNAs) are involved in disease pathogenesis, and the roles of lncRNAs during pancreatic carcinogenesis remain poorly understood. Some lncRNAs are found in extracellular vesicles (EVs); EV-encapsulated RNAs in serum (obtained via liquid biopsy) have been used as biomarkers. We previously verified that the lncRNA highly upregulated in liver cancer (HULC) promotes invasion and migration by regulating the epithelial-mesenchymal transition (EMT) via EV transfer in pancreatic ductal adenocarcinoma (PDAC) cells. However, little is known about whether “EV lncRNAs” could serve as biomarkers or regulators of tumor growth in PDAC.

Aims & Methods

We aimed to identify novel EV lncRNAs that serve as serum biomarkers for PDAC and to investigate the functional roles mediated by lncRNAs in PDAC development. EV RNA was isolated from the serum of 3 PDAC patients and 3 healthy individuals. Gene expression in the serum EVs was examined using the Affymetrix cDNA microarray. LncRNA expression levels and functions were assessed using human pancreatic ductal epithelial (HPDE) and PDAC (e.g., Panc-1) cells. siRNAs and lentiviral vectors were used to modulate RNA expression. Cell invasion, migration, and apoptosis were examined by transwell, wound healing, and caspase 3/7 assays. Expression of RNA was assessed by qPCR and of protein by Western blot. Serum samples were collected from patients with PDAC (n=22) and intraductal papillary mucinous neoplasms (IPMNs; n=23) and healthy individuals (n=21). EV lncRNA expression in serum was analyzed by digital PCR. The diagnostic accuracy of various markers was evaluated using receiver operating characteristic (ROC) analysis, with measurement of the area under the curves (AUC) values.

Results

Gene expression analyses detected 1,205 genes that exhibited two-fold differential expression; the levels of 1,069 increased, and those of 136 decreased, in serum EVs of PDAC patients compared to those of healthy individuals. Of these lncRNAs, LINC02280 and LOC100507412 (termed HEVEPA) were the most significantly upregulated; both were highly expressed in a panel of PDAC relative to HPDE cells. In Panc-1 cells, knockdown of LINC02280 by siRNA significantly increased expression of E-cadherin and decreased expression of N-cadherin, Snail, and Vimentin, and cell invasion and migration. Moreover, overexpression of LINC02280 significantly reduced the expression of E-cadherin and increased the expression of N-cadherin, Snail, and Vimentin, and cell invasion and migration. Altogether, LINC02280 promoted cell invasion and migration through the induction of EMT. Furthermore, we found that HEVEPA inhibited the apoptosis of Panc-1 cells. These observations suggested that LINC02280 and HEVEPA would be oncogenic. In serum EVs, the expression levels of LINC02280, HEVEPA, and HULC were significantly elevated in PDAC patients compared to healthy individuals and IPMN patients (p < 0.05). The AUC values of the combinations [LINC02280, HEVEPA, HULC, and CA19-9] and [LINC02280, HEVEPA, and HULC] were higher than those of LINC02280, HEVEPA, HULC, or CA19-9 alone (1.00 and 0.98 vs. 0.89, 0.80, 0.92, and 0.89, respectively) in terms of discriminating PDAC from healthy individuals and IPMN patients.

Conclusion

LINC02280 and HEVEPA promote tumor development in PDAC. A combination of EV lncRNAs and CA19-9 showed good performance in predicting PDAC diagnosis and will find applications in liquid biopsies to determine human PDAC status.

Introduction

Type 1 diabetes is a disease of the endocrine pancreas, but it also affects the exocrine part, especially the ductal cells. Several studies have shown that secretin-induced ductal HCO₃^- secretion is reduced in diabetes, however, the underlying mechanism and the effect of diabetes on basal secretion is less known.

Aims & Methods

Our aim is to investigate the effect of diabetes on pancreatic exocrine function. Diabetes was induced in wild type and cystic fibrosis transmembrane conductance regulator (CFTR) knock out mice by i.p. administration of streptozotocin and disease development was confirmed by fasting blood glucose level measurement. Pancreatic ductal fluid and HCO₃^- secretion were measured by fluid secretion measurements and fluorescence microscopy, respectively. Expression of ion transporters were investigated by real-time PCR and immunohistochemistry, whereas TEM was used for the morphological characterization of the pancreas.

Results

​Basal fluid and HCO₃^- secretion are significantly elevated in diabetes. Acute or chronic glucose treatment did not affect HCO₃^- secretion, but inhibition of CFTR significantly reduced it in both normal and diabetic mice. Cl^- efflux and the expression of CFTR, ANO1, NHE-1 and AQP1 increased in diabetes. Secretin-stimulated fluid secretion was also significantly higher in diabetic mice.

Conclusion

Our results show that diabetes increases fluid and HCO₃^- secretion in ductal cells both under basal and stimulated conditions in which the increased function of ion and water transporters, especially CFTR, plays central role.
This study was supported by the National Research, Development and Innovation Office Grant (SNN134497 to VV) and the CFRD-SRC Grant (No.: SRC 007).