Islet neogenesis-associated protein-related pentadecapeptide enhances the differentiation of islet-like clusters from human pancreatic duct cells Juan Li a,1 , Yun Wang a,1 , Xiaozhu Yu a,1 , Haiyan Chen a , Ying Wu a , Xiao Han b,c , Xirong Guo d , Chenyu Zhang b,e , Qi Chen b,c , Jiawei Chen a,b , Tao Yang a,b, * a Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, 210029, Jiangsu, China b Jiangsu Diabetes Center, 300 Guangzhou Road, Nanjing, 210029, Jiangsu, China c Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, Jiangsu, China d Institute of Pediatrics of Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, Jiangsu, China e State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing, 210093, Jiangsu, China 1. Introduction Islet transplantation is a promising possibility for the optimal treatment of type 1 and type 2 diabetes [19]. Successful islet transplantation has the potential to achieve physiologic glucose control, thereby reducing or eliminating the need for daily medication as well as preventing long-term diabetic complications [13]. However, one of the major obstacles to successful islet transplantation is the limitation of available insulin-producing tissue. The Edmonton Protocol [21] requires the utilization of islets from two to three donor pancreata for successful transplantation, resulting in insulin independence for a single patient. The need for transplantable human islets has stimulated efforts to expand existing pancreatic islets and/or to develop an abundant in vitro source of human insulin-producing cells [2]. Various sources of insulin-producing tissue have been proposed including porcine tissue, engineered b-cell lines and embryonic stem cells, each with a non-ideal differentiation rate [12]. Based on studies on rat and human pancreatic regeneration, we have concluded that adult pancreatic duct cells have the capacity to expand and differentiate. However, the amounts of insulin made by b-cells differentiated from these cells were miniscule compared to the amount made by native islets. Islet neogenesis-associated protein (INGAP), originally named ilotropin [16], was first found in the pancreas head of normal hamsters previously wrapped in cellophane [16,27]. INGAP’s mRNA was detected in exocrine pancreatic cells and INGAP transcription was also identified in duct cells and islet non-b-cells [3,8]. A pentadecapeptide having the 104–118 amino acid sequence of INGAP (INGAP-PP) reproduced the stimulatory effect of the intact molecule upon thymidine Peptides 30 (2009) 2242–2249 ARTICLE INFO Article history: Received 6 April 2009 Received in revised form 25 August 2009 Accepted 2 September 2009 Available online 10 September 2009 Keywords: Islet neogenesis INGAP Differentiation Stem cells ABSTRACT The differentiation of pancreatic ductal epithelial cells into b-cells has been considered as an alternative method for increasing the number of islets for transplantation. Critical factors have been introduced into the in vitro differentiation protocol for pancreatic duct cells in order to enhance the production of b-cells. Islet neogenesis-associated protein (INGAP) is an initiator of islet neogenesis and the peptide sequence 104–118 of INGAP has been shown to stimulate an increase in b-cell mass in animals and also found in human pathological states involving islet neogenesis. To establish a novel method for the differentiation of b-cells from human pancreatic duct cells with INGAP-related pentadecapeptide (INGAP-PP), the pancreatic duct cells were isolated, purified and expanded in vitro and differentiated using a four-step protocol that included nicotinamide, exendin-4, transforming growth factor b 1 and INGAP-PP/ Scrambled peptide (Scrambled-P). The production of islet-like clusters (ILCs) in the INGAP-PP group was significantly higher than that in the Scrambled-P control group after differentiation from an equal number of duct cells. The duct cells showed positive staining and expression for cytokeratin 19, pancreatic duodenal homeobox-1, nestin, and were negative for insulin and glucagon, as detected by both immunofluorescence and RT-PCR. Following differentiation the cells became insulin and glucagon positive. In addition, the ILCs from the INGAP-PP group secreted higher levels of insulin and C-peptide than the Scrambled-P group under a high glucose challenge. We conclude that INGAP peptide enhances the in vitro differentiation of pancreatic duct cells into islet-like clusters. ß 2009 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +86 25 83781781; fax: +86 25 83781781. E-mail address: yangt@njmu.edu.cn (T. Yang). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Peptides journal homepage: www.elsevier.com/locate/peptides 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.09.003