Lineage tracing of pancreatic exocrine cell plasticity

Abstract

Cell lineage tracing is an important tool to provide direct evidence for the origin and fate of cells. Using this tool, cells can be indelibly labelled to identify their progeny during normal development as well as under various experimental conditions. Therefore, lineage tracing allows for a better characterization of progenitor cells. Improvement of this fundamental knowledge could help to find new sources of replacement tissues to treat degenerative diseases such as diabetes mellitus. The current treatments for diabetes cannot prevent its long-term complications. Beta cell therapy is promising, however its application is currently limited by donor shortage. The beta cell mass can be increased under several conditions, however the exact origin of the newly formed beta cells is usually unclear. A better understanding of the beta cell mass expansion and of the cellular plasticity in the pancreas is therefore of utmost importance since this could lead to the development of novel methods of generating novel beta cells. Here, we developed and applied methods for cell lineage tracing to study pancreatic plasticity.
In the first part of this work, we performed lineage tracing in transgenic Hnf1b-CreERT R26R reporter mice to answer the longstanding unsettled question of whether beta cells originate from exocrine duct cells (in collaboration with the lab of Prof. Dr. Jorge Ferrer from Barcelona, Spain). Hnf1b is identified as a specific ductal marker, both in the developing and adult pancreas. We demonstrate that Hnf1b positive cells of the early embryonic ducts are precursors of acinar, ductal and endocrine lineages. Later during embryonic development Hnf1b positive cells only give rise to duct and endocrine cells, but not to acinar cells. In addition, the ductal epithelium does not significantly contribute to acinar or endocrine cells during neonatal growth, postnatal development or during beta cell growth triggered by partial duct ligation or by beta cell-specific ablation with alloxan followed by epidermal growth factor/gastrin treatment.
In the second part, we focussed on acinar cell plasticity. The plasticity of rodent acinar cells both in vitro and in vivo has already been extensively documented. Acinar cells represent by far the largest component of the pancreas, thereby forming an attractive target for cell reprogramming to generate new beta cells. However, little is known about the plasticity of human acinar cells. In this work we present pioneering tracing experiments to study the fate of human acinar cells in culture. We demonstrate, both with genetic and non-genetic lineage tracing, that human acinar cells spontaneously transdifferentiate to duct cells. Within one week virtually all surviving acinar cells had adopted a ductal phenotype.
Since our findings on human cells and previous studies in rodents indicated significant plasticity of pancreatic exocrine acinar cells, establishment of an efficient method for gene delivery in pancreatic acinar cells would represent a useful tool to further unravel the underlying mechanisms via gain- and loss-of-function studies and for tracing. Hence, different transfection/transduction methods were evaluated for their gene delivery efficiency in pancreas, both in vitro and in vivo. We demonstrate that VSV-G pseudotyped lentiviral vectors provide the best efficiency, combined with optimal cell viability for in vitro gene delivery to exocrine pancreas cells. However, transduction of rodent pancreas in vivo by intraparenchymal injection of lentiviral vectors proved to be inefficient. Therefore, an in vivo adenoviral transduction method for rodent pancreas, resulting in high transduction efficiencies, was developed.
Taken together, our findings may have important consequences for developing novel therapeutic strategies for diabetes and pancreatic cancer since they contribute to enhance our understanding of pancreatic plasticity. In addition, the tracing and transduction methods that we developed in this work will undoubtedly prove to be helpful in future studies of (human) acinar cell transdifferentiation/reprogramming.

Date of Award	19 Sept 2011
Original language	English
Awarding Institution	Vrije Universiteit Brussel
Supervisor	Luc Bouwens (Promotor), Henry Heimberg (Co-promotor), Karin Vanderkerken (Jury), Pieter in 't Veld (Jury), Ivan Van Riet (Jury), Pedro Luis Herrera (Jury), André Herchuelz (Jury) & Frans Schuit (Jury)