Establishment of a high-resolution 3D modeling system for studying pancreatic epithelial cell biology in vitro

Mostafa Bakhti, Katharina Scheibner, Sophie Tritschler, Aimée Bastidas-Ponce, Marta Tarquis-Medina, Fabian J. Theis, Heiko Lickert

Over the past decade, our understanding of human pancreas development has steadily increased. This is partially due to the recent advancements in differentiation of human pluripotent stem cells into pancreatic islet-like clusters. Although this in vitro differentiation system has provided lots of information, it cannot address the impact of tissue morphogenesis on endocrine cell differentiation. Therefore, Bakhti, Scheibner, et al. developed a simple ex vivo 3D cyst culture from pancreatic progenitors, providing a high-resolution modeling system that not only allows for studying pancreas development in a temporal fashion but also enables comparing pancreatic epithelial biology across species and genotypes.

Objective: Translation of basic research from bench-to-bedside relies on a better understanding of similarities and differences between mouse and human cell biology, tissue formation, and organogenesis. Thus, establishing ex vivo modeling systems of mouse and human pancreas development will help not only to understand evolutionary conserved mechanisms of differentiation and morphogenesis but also to understand pathomechanisms of disease and design strategies for tissue engineering.

Methods: Here, we established a simple and reproducible Matrigel-based three-dimensional (3D) cyst culture model system of mouse and human pancreatic progenitors (PPs) to study pancreatic epithelialization and endocrinogenesis ex vivo. In addition, we reanalyzed previously reported single-cell RNA sequencing (scRNA-seq) of mouse and human pancreatic lineages to obtain a comprehensive picture of differential expression of key transcription factors (TFs), cell–cell adhesion molecules and cell polarity components in PPs during endocrinogenesis.

Results: We generated mouse and human polarized pancreatic epithelial cysts derived from PPs. This system allowed to monitor establishment of pancreatic epithelial polarity and lumen formation in cellular and sub-cellular resolution in a dynamic time-resolved fashion. Furthermore, both mouse and human pancreatic cysts were able to differentiate towards the endocrine fate. This differentiation system together with scRNA-seq analysis revealed how apical-basal polarity and tight and adherens junctions change during endocrine differentiation.

Conclusions: We have established a simple 3D pancreatic cyst culture system that allows to tempo-spatial resolve cellular and subcellular processes on the mechanistical level, which is otherwise not possible in vivo.