JOP. J Pancreas (Online) 2008; 9(5):601-611. JOP. Journal of the Pancreas - http://www.joplink.net - Vol. 9, No. 5 - September 2008. [ISSN 1590-8577] 601 ORIGINAL ARTICLE Assessment of in Vitro Differentiation of Bovine Pancreatic Tissue in Insulin-Expressing Cells Marina Figliuzzi 1 , Federica Adobati 1 , Roberta Cornolti 1 , Paola Cassis 1 , Giuseppe Remuzzi 1,2 , Andrea Remuzzi 1,3 1 Department of Biomedical Engineering, Mario Negri Institute for Pharmacological Research. Bergamo, Italy. 2 Unit of Nephrology and Dialysis, Ospedali Riuniti di Bergamo. Bergamo, Italy. 3 Department of Industrial Engineering, University of Bergamo. Dalmine (BG), Italy ABSTRACT Context Expansion and culture of beta cell progenitors in vitro may represent an alternative to the use of differentiated beta cells from donor pancreata. Objective The aim of our study was to investigate to what extent exocrine or endocrine pancreatic cells can be differentiated in insulin-producing cells in vitro. Setting Bovine exocrine tissue (n=4) and islets (n=4) were cultured in DMEM with serum. Interventions After 7 days, the cells were trypsinized and cultured in the same medium for cell proliferation, or in DMEM/F-12 containing growth factors to induce cell differentiation. Main outcome measure Proliferating capacity after 4 weeks in culture. In addition, insulin expression was evaluated by RT-PCR and by immunohistochemical staining. Results After 4 weeks of culture, cells from exocrine tissue showed a 69.5±10.0 fold increase, while cells from islets showed a 31.2±11.4 fold increase (P=0.059). In differentiating medium, monolayers from exocrine and islet tissue were organized into islet-like structures containing cells which stained positively for insulin. Morphometrical analysis and RT-PCR confirmed the presence of insulin in the cells at the protein and the mRNA level. Conclusions In our experimental conditions, cells from pancreatic tissue proliferated and differentiated in insulin-containing cells. However, the level of insulin as well as mRNA expression is only a small fraction of that shown by fresh islets. Only selective identification of cell precursors may allow efficient generation of insulin-producing cells in vitro. INTRODUCTION Replacement of the damaged pancreatic beta cells in insulin-dependent diabetic patients represents a strategy for restoring glycemic control. Among others, the most severe limitations of this approach are the scarcity of organ donation with respect to the large number of diabetic patients. New strategies for obtaining insulin producing cells from other sources are under extensive investigation. Beta cells in the adult pancreas have a brief life span after which they undergo apoptosis [1]. It has been documented that senescent beta cells are replaced by the replication of existing mature beta cells [2] and also by the differentiation or neogenesis of beta cells deriving from exocrine or intra-islet pancreatic precursor cells [3, 4, 5].