Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells

Jonghyeob Lee,Satsuki Miyazaki,Gregory L Szot,Seung K Kim,James F Markmann,Ji Lei,Xueying Gu,Jing Wang,Yinghua Liu,Takuya Sugiyama,Rita Bottino,Jun-Ichi Miyazaki

doi:10.7554/elife.00940

Abstract

Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.

Highlights

The pancreas is a vital organ with exocrine and endocrine cell functions, and a root of lethal human diseases including diabetes mellitus, pancreatitis, and pancreatic ductal adenocarcinoma
To identify human pancreatic epithelial cells that can be grown and maintained in culture, we systematically screened cell isolation methods and culture conditions with dispersed adult human pancreatic cells obtained from cadaveric donors without known pancreatic cancer, diabetes mellitus, or other pancreatic diseases (Table 1)
A survey of cell surface markers used for fetal mouse pancreatic cell isolation (Sugiyama et al, 2007) revealed that antibodies recognizing CD133 enriched sphere-forming cells by four fold, whereas sphere-forming cells were depleted in the CD133neg fraction (Figure 1A,B)

Summary

Introduction

The pancreas is a vital organ with exocrine and endocrine cell functions, and a root of lethal human diseases including diabetes mellitus, pancreatitis, and pancreatic ductal adenocarcinoma. Pancreatic endocrine functions derive from clusters of epithelial cells (islets of Langerhans) called α-, β-, δ-, and PP-cells that respectively synthesize, store, and secrete the hormones Glucagon, Insulin, Somatostatin, and Pancreatic polypeptide (Benitez et al, 2012). Insulin production by islet β-cells is highly regulated: key features of mature β-cells include preproinsulin (INS) transcription, proinsulin processing by endo- and exopeptidases and storage of the proinsulin cleavage products insulin and C-peptide in dense core vesicles. Cardinal β-cell functions regulate insulin release in response to glucose and other secretagogues, including glucose sensing and metabolism through the enzyme glucokinase, and use of ATP-dependent potassium channels (KATP) and voltage-gated calcium channels to induce insulin

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Results

Conclusion