Directed Pancreatic Acinar Differentiation of Mouse Embryonic Stem Cells via Embryonic Signalling Molecules and Exocrine Transcription Factors

Pluripotent embryonic stem cells (ESC) are a promising cellular system for generating an unlimited source of tissue for the treatment of chronic diseases and valuable in vitro differentiation models for drug testing. Our aim was to direct differentiation of mouse ESC into pancreatic acinar cells, which play key roles in pancreatitis and pancreatic cancer. To that end, ESC were first differentiated as embryoid bodies and sequentially incubated with activin A, inhibitors of Sonic hedgehog (Shh) and bone morphogenetic protein (BMP) pathways, fibroblast growth factors (FGF) and retinoic acid (RA) in order to achieve a stepwise increase in the expression of mRNA transcripts encoding for endodermal and pancreatic progenitor markers. Subsequent plating in Matrigel® and concomitant modulation of FGF, glucocorticoid, and folllistatin signalling pathways involved in exocrine differentiation resulted in a significant increase of mRNAs encoding secretory enzymes and in the number of cells co-expressing their protein products. Also, pancreatic endocrine marker expression was down-regulated and accompanied by a significant reduction in the number of hormone-expressing cells with a limited presence of hepatic marker expressing-cells. These findings suggest a selective activation of the acinar differentiation program. The newly differentiated cells were able to release α-amylase and this feature was greatly improved by lentiviral-mediated expression of Rbpjl and Ptf1a, two transcription factors involved in the maximal production of digestive enzymes. This study provides a novel method to produce functional pancreatic exocrine cells from ESC. © 2013 Delaspre et al.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII)-FEDER (PI052738 and PI080511 to A.S.; PI100094 and Tercel RD06/0010/ 0025 to B.S.). A.S. was supported by ISCIII and the Health Department of the Generalitat de Catalunya; F.D. was the recipient of a Graduate Fellowship from ISCIII and was also supported by Tercel; M.M. was the recipient of a Graduate Fellowship from the Generalitat de Catalunya.Peer Reviewe

Centroacinar cells are progenitors that contribute to endocrine pancreas regeneration

Diabetes is associated with a paucity of insulin-producing β-cells. With the goal of finding therapeutic routes to treat diabetes, we aim to find molecular and cellular mechanisms involved in β-cell neogenesis and regeneration. To facilitate discovery of such mechanisms, we use a vertebrate organism where pancreatic cells readily regenerate. The larval zebrafish pancreas contains Notch-responsive progenitors that during development give rise to adult ductal, endocrine, and centroacinar cells (CACs). Adult CACs are also Notch responsive and are morphologically similar to their larval predecessors. To test our hypothesis that adult CACs are also progenitors, we took two complementary approaches: 1) We established the transcriptome for adult CACs. Using gene ontology, transgenic lines, and in situ hybridization, we found that the CAC transcriptome is enriched for progenitor markers. 2) Using lineage tracing, we demonstrated that CACs do form new endocrine cells after β-cell ablation or partial pancreatectomy. We concluded that CACs and their larval predecessors are the same cell type and represent an opportune model to study both β-cell neogenesis and β-cell regeneration. Furthermore, we show that in cftr loss-of-function mutants, there is a deficiency of larval CACs, providing a possible explanation for pancreatic complications associated with cystic fibrosis

Nano-Sampling and Reporter Tools to Study Metabolic Regulation in Zebrafish

In the past years, evidence has emerged that hallmarks of human metabolic disorders can be recapitulated in zebrafish using genetic, pharmacological or dietary interventions. An advantage of modeling metabolic diseases in zebrafish compared to other “lower organisms” is the presence of a vertebrate body plan providing the possibility to study the tissue-intrinsic processes preceding the loss of metabolic homeostasis. While the small size of zebrafish is advantageous in many aspects, it also has shortcomings such as the difficulty to obtain sufficient amounts for biochemical analyses in response to metabolic challenges. A workshop at the European Zebrafish Principal Investigator meeting in Trento, Italy, was dedicated to discuss the advantages and disadvantages of zebrafish to study metabolic disorders. This perspective article by the participants highlights strategies to achieve improved tissue-resolution for read-outs using “nano-sampling” approaches for metabolomics as well as live imaging of zebrafish expressing fluorescent reporter tools that inform on cellular or subcellular metabolic processes. We provide several examples, including the use of reporter tools to study the heterogeneity of pancreatic beta-cells within their tissue environment. While limitations exist, we believe that with the advent of new technologies and more labs developing methods that can be applied to minimal amounts of tissue or single cells, zebrafish will further increase their utility to study energy metabolism