Differentiation of Affinity-Purified Human Pancreatic Duct Cells to β-Cells

Abstract

To test whether pancreatic duct cells are in vitro progenitors, they were purified from dispersed islet-depleted human pancreatic tissue using CA19-9 antibody. The purified fraction was almost entirely CK19؉ with no insulin ؉ cells, whereas the unpurified cells (crude duct) were 56% CK19 W hereas islet transplantation is an effective and beneficial treatment for type 1 diabetes, its application is limited by the shortage of islets. A possible solution is to generate insulin-producing cells from adult stem/progenitor cells of the pancreas. In vivo new ␤-cells are generated through replication of preexisting ␤-cells and neogenesis, the latter from differentiation of non-hormone-expressing progenitor cells (1-8). Putative adult stem/progenitor cells from mouse pancreas have been expanded clonally and after manipulation were found to express low levels of insulin and other pancreatic markers (9,10). While these findings are provocative, it has not yet been shown that such cells can become fully functional ␤-cells (11).Our group reported that islet-like structures, which secrete insulin in response to glucose, could be generated from islet-depleted pancreatic tissue remaining after human islet isolation (12). These findings were confirmed and extended by Otonkoski and colleagues (13). The cell of origin has been suspected to be ductal in origin but has not been conclusively shown. Additionally, three other groups (14 -16) have reported that putative progenitor cells, which arose from human islet preparations, could be expanded through many passages and then be manipulated to reexpress islet hormones at low levels. Gershengorn et al. (16), Habener and colleagues (14,17) and Efrat and colleagues (18) have suggested that the expanding cells are ␤-cells that have undergone epithelial-mesenchymal transition, no longer express insulin, and have great capacity for expansion. However, even the purest human islet preparations are not pure islet cells but contain many contaminating duct, acinar, and connective tissue cells. Olson and colleagues (15) showed that serpinginous cells expressing vimentin and nestin had no islet hormones during expansion but acquired low levels of islet markers after manipulation of culture conditions. Similar cells were initially suggested to be the preexisting nestinpositive cells found in the islets and in the ductal stroma (17). All of these studies have raised the possibility of generating new islet cells in vitro from human pancreatic tissue, but in each case the cell of origin has not been identified; thus, it is not clear whether ␤-cells actually undergo such a transition.The purpose of this study is to test whether highly purified human adult pancreatic duct cells can differentiate in vitro into insulin-producing cells. To this end, extremely pure duct preparations were obtained following immunomagnetic sorting with CA19-9 antibody. The affinity purification step is highly selective for pancreatic duct epithelial cells and is performed immediately after islet purification. Our method is qu...

A simple two-step protocol for the purification of human pancreatic beta cells

“…However, the use of CA19-9 microbeads allows much more efficient purification of ductal cells, as described previously [9,11]. Using the combination of microbeads targeted against CA19-9 and PSA-NCAM, it is now possible to isolate highly purified populations of ductal and beta cells in a simple two-step procedure.…”

“…Negative cell fraction was immediately used for PSA-NCAM sorting as described above. All procedures of cell sorting except incubations were done at room temperature to minimise cell death [9].…”

“…The method is based on the elimination of ductal cells using an antibody against carbohydrate antigen 19-9 (CA19-9), an antigen expressed on the luminal surface of pancreatic ducts [8,9], followed by positive selection of cells expressing PSA-NCAM.…”

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

“…The population of ductal cells used in the present investigation has previously been described [21,24,28]. Thus, the expansion of cells derived from the islet-depleted exocrine fraction, which is usually discarded following isolation, has been shown to give rise to a near homogeneous population of CK19-positive epithelial cells, with minimal fibroblast contamination during the initial phase of culture [21,25].…”

A role for islet neogenesis in curing diabetes

“…This system has been adapted to measure islet neogenesis as a pharmacological activity as well as to elucidate the provenance of islet tissue [18,28]. Application of growthpromoting substances to ductal cells in culture have stimulated these to grow and express PDX1, which encodes the transcription factor necessary for endocrine cell development [29,30]. At this stage, adding matrigel with a switch to a medium containing keratinocyte growth factor and nicotinamide induces the cells to form cultivated human islet buds that produce a small amount of insulin in response to glucose.…”