Use of Live Murine Pancreatic Slices for the Study of ß-Cell Regeneration

Abstract

Introduction: Bone morphogenetic protein 7 (BMP-7), a protein with BMP-activating and TGF-β inactivating properties, induces the proliferation of a unique population of progenitor-like cells characterized by the expression of PDX1 and the BMP receptor activin-like kinase 3 (ALK3/BMPR1A). These cells can be sorted using ALK3 and the purinergic receptor P2Y1 (P2RY1) as a novel surrogate surface marker for PDX1. Sorted P2RY1+/ALK3bright+ cells form BMP-7 expandable colonies. Upon BMP-7 withdrawal, they differentiate into cells of all three lineages (endocrine, acinar and ductal) of the adult pancreas. PDX1+/ALK3+ cells absent from islets are represented in the major pancreatic ducts and pancreatic duct glands. Preliminary evidence indicates that these cells are also present in the murine pancreas. We hypothesized that islet β-cell regeneration could be studied in real time using live murine pancreatic slices transduced with a loxP-dsRed-loxP-EGFP reporter + insulin promoter-Cre adenovirus. If new β-cells arise from preexisting insulin-expressing-cells they would remain green. However, if neogenesis occurs from non-β-cells, there is a window of time during which the degrading red and the new green protein co-localize in the cell. Allowing us to discriminate between neogenesis and β-cell proliferation. Methods: Low melting point (LMP) agarose-embedded, vibratome-sectioned live pancreatic slices were generated from CD-1 mice. Transduction with a loxP-dsRed-loxP-EGFP reporter + insulin promoter-Cre adenovirus was done in slices cultured atop optically clear membranes. Slices were treated for 4 days with an ALK3 agonist (THR-123) or saline at the same time of the co-transduction. Bright field and fluorescence images of them were taken every 24h for 7 days. Conclusions: This is the first report on the use of live pancreatic slices as a novel tool for the study of pancreatic regeneration. Our preliminary results suggest that there are quantifiable β-cell neogenic events in response to BMP-7-like agents. Disclosure F. Qadir: None. J. Weitz: None. S. Alvarez-Cubela: None. D. Klein: None. G. Lanzoni: None. C.A. Garcia-Santana: Employee; Self; Ophysio Incorporated. A. Montalvo: None. C. Ricordi: None. L. Inverardi: None. A. Caicedo: None. R. Pastori: None. J. Domínguez-Bendala: Stock/Shareholder; Self; Ophysio.