Neonatal Pig Liver—Derived Progenitors for Insulin-Producing Cells: An In Vitro Study LEDA RACANICCHI, GIUSEPPE BASTA, PIA MONTANUCCI, LUCIA GUIDO, ALESSANDRA PENSATO, VALENTINA CONTI, and RICCARDO CALAFIORE ABSTRACT Beta (b)-cell replacement represents an attractive approach for the possible cure of type 1 insulin- dependent diabetes mellitus (IDDM). In a search for potential sources of insulin-secreting cells for IDDM substitution therapy, we have focused on the neonatal pig liver, which is putatively enriched in multi- potent stem cells. We then isolated cells measuring 10 to 15 lm in diameter, identified as small cells, characterized by a high proliferation rate and positive staining for immature liver and pancreatic endo- crine cell markers (i.e., insulin and pancreatic duodenal homeobox). The ability of these cells to trans- differentiate into pancreatic b-like cells under culture conditions with exendin-4 (Ex-4) or high glucose concentration was examined. We observed that insulin secretion was not physiological in basal condi- tions, although it became responsive to glucose after 5 days of exposure to Ex-4. This b-cell-like phe- notype remained physiologically stable, even after stimulus withdrawal. Based on these observations, we contend that the proposed cell and tissue model might offer several advantages as a candidate for sub- stitution cell therapy in IDDM, because the neonatal pig liver seems enriched in cells, with a mixed pancreas–liver phenotype, that are easier to purify and grow in culture and are more functional than other b-like cells upon in vitro single short-term stimulation challenge. INTRODUCTION T HE RESTRICTED AVAILABILITY OF cadaveric human donor pancreases as a source of beta (b)-cells strictly ham- pers progress of clinical trials of cell therapy to treat type 1 insulin-dependent diabetes mellitus (IDDM). Inducing dif- ferentiation of other cell lineages toward endocrine cell phe- notypes might surmount this hurdle. In a search for these potential sources of b-like cells, it has been reported that adult or fetal stem cells, 1 because of their plasticity and high proliferation rate, could be suitable for this purpose. However, although fetal cells are associated with a series of problems that require resolution (e.g., teratogenity), 2 adult cells would be safer for in vivo application if their number per organ cell mass was not minimal. Organ-specific stem cells have been identified in the bone marrow, liver, and brain, but not in the pancreas, 3 with few exceptions. 4 Many authors have postulated that the ‘‘pan- creatic stem cell’’ does not exist and that regenerating b- cells observed in particular conditions (e.g., pregnancy, obesity) could derive from duplication of existing b-cells or differentiating precursors. 5 Nevertheless, a cell population hallmarked by small diameter has been recently identified in the pancreas that might represent a prototype of ‘‘pan- creatic stem or progenitor cell.’’ 6,7 At this juncture, no multipotent cells, to be expanded and differentiated in vitro into insulin-secreting cells, may be retrieved in bulk from the pancreas. To avoid these restrictions, the liver could serve as an alternative cell source for insulin producing or releasing cells, because of the common embryonic origin and the similar glucose-sensing system 8 of the pancreas and liver. Transdifferentiation of hepatic stem cells, 9 human fetal liver cells, 10,11 and human adult hepatocytes 12 into functional Department of Internal Medicine, Section of Internal Medicine and Endocrine and Metabolic Sciences, University of Perugia, Perugia, Italy. TISSUE ENGINEERING Volume 00, Number 00, 2007 # Mary Ann Liebert, Inc. DOI: 10.1089/ten.2007.0098 1 (page numbers are temporary)