Enriched Human Pancreatic Ductal Cultures Obtained from Selective Death of Acinar Cells Express Pancreatic and Duodenal Homeobox Gene-1 Age-Dependently

Abstract

Adult pancreatic ductal cells are believed to be islet precursors. Our aim was to obtain an enriched human ductal cell population in defined culture conditions, and to characterize these cultures for the presence of pancreatic developmental transcription factors. Non-endocrine adult human pancreatic digest was cultured for 4 days in serum-containing and serum-free media. During this time, analysis was done for phenotypic changes, cell death, and expression of islet and islet precursor markers. Culture in serum-supplemented and serum-free media gave similar recoveries of an enriched ductal population after 4 days. Extensive cell death due to apoptosis and necrosis was also observed over this time period. A donor-age dependent expression of pancreatic and duodenal homeobox gene-1 (PDX-1) in ductal cells was seen at 4 days whereby donors <25 yr expressed significantly more than donors >25 yr. Analysis of gene expression by RT-PCR showed the presence of islet developmental transcription factors neuroD, Nkx6.1, and PDX-1, as well as mature islet hormones. While acinar-ductal transdifferentiation of some cells cannot be ruled out, we provide evidence that the predominant mechanism for the derivation of enriched human ductal cultures in our culture conditions is selective acinar cell death. Furthermore, we have shown that ductal cultures from younger donors exhibit greater plasticity through expression of PDX-1, and may be of greater value in attempts to induce islet neogenesis. The presence, however, of insulin and glucagon mRNA indicates that contaminating endocrine cells remain in these cultures and underscores the need to use caution when assessing differentiation potential.

The impact of the mTOR inhibitor sirolimus on the proliferation and function of pancreatic islets and ductal cells

“…Medium was exchanged at day 1 post culture and every 2 days thereafter. At the end of the culture period these preparations are composed of 68% cytokeratin (CK)-19, 16% amylase, and 1% beta cells [31].…”

“…After isolation and 4 to 6 days of culture [31] HDCs were cultured at ∼1.5×10 5 cells/well with and without 10 or 20 ng/ml sirolimus (Rapamune; Wyeth-Canada Laboratories, Markham, ON, Canada). NPIs (535 aggregates/well) were cultured as experimental cultures with 20 ng/ml of sirolimus added to the base media or as control cultures grown in base media alone.…”

“…Cell preparations were assessed for cell number by measuring DNA content at time 0 and day 6 of culture. We have previously shown that human islet cells contain 6.6 pg DNA/cell [31] whereas NPI cells have 7.1 pg DNA/cell [26]. Cellular insulin content and cellular composition of the NPI cultures were also characterised at these timepoints.…”

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium

“…3,6,10,11 Recently, we have described an in vitro method that enriches the ductal-epithelial cell fraction while depleting the acinar component of the pancreatic exocrine tissue following human islet purification. 12 However, in our culture conditions, we had been unable to expand insulin-producing cells from this enriched ductal cell fraction. Interestingly, we have observed an outgrowth of adherent 'fibroblast-like', spindleshaped cells from the ductal-epithelial cell aggregates, which we characterize in the current study as mesenchymal stem cells (MSCs).…”

Co-encapsulation of bioengineered IGF-II-producing cells and pancreatic islets: effect on beta-cell survival

“…The beneficial effect of pancreatic duct cells, which produce IGF-II, on islet cell survival has also been supported by the finding of a significant correlation between a higher percentage of duct cell contamination in islet preparations and a positive outcome of clinical transplantations. 8 The mechanisms underlying these observations are unknown. Such an association has not been found with any other components of the pancreas.…”