Published online Feb 15, 2014. doi: 10.4239/wjd.v5.i1.59
Revised: November 27, 2013
Accepted: December 13, 2013
Published online: February 15, 2014
AIM: To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm that β-cell progenitors reside within the pancreatic epithelium.
METHODS: Due to mesenchymal stem cell (MSC) expansion and overgrowth, progenitor cells within the pancreatic epithelium cannot be characterized in vitro, though β-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition (EMT) may generate β-cell progenitors. Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth (dexamethasone, epidermal growth factor, 3,5,3’-triiodo-l-thyronine, bovine brain extract). Cells were also infected with a single and dual lentiviral reporter prior to cell differentiation. Enhanced green fluorescent protein was controlled by the rat Insulin 1 promoter and the monomeric red fluorescent protein was controlled by the mouse PDX1 promoter. In combination with lentiviral tracing, cells expanded and differentiated in the pancreatic medium were characterized by flow cytometry (BD fluorescence activated cell sorting), immunostaining and real-time polymerase chain reaction (PCR) (7900HT Fast Realtime PCR System).
RESULTS: In the presence of 10% serum MSCs rapidly expand in vitro while the epithelial cell population declines. The percentage of vimentin+ cells increased from 22% ± 5.83% to 80.43% ± 3.24% (14 d) and 99.00% ± 0.0% (21 d), and the percentage of epithelial cells decreased from 74.71% ± 8.34% to 26.57% ± 9.75% (14 d) and 4.00% ± 1.53% (21 d), P < 0.01 for all time points. Our novel pancreatic epithelial expansion medium preserved the epithelial cell phenotype and minimized epithelial cell dedifferentiation and EMT. Cells expanded in our epithelial medium contained significantly less mesenchymal cells (vimentin+) compared to controls (44.87% ± 4.93% vs 95.67% ± 1.36%; P < 0.01). During cell differentiation lentiviral reporting demonstrated that, PDX1+ and insulin+ cells were localized within adherent epithelial cell aggregates compared to controls. Compared to starting islets differentiated cells had at least two fold higher gene expression of PDX1, insulin, PAX4 and RFX (P < 0.05).
CONCLUSION: PDX1+ cells were confined to adherent epithelial cell aggregates and not vimentin+ cells (mesenchymal), suggesting that EMT is not a mechanism for generating pancreatic progenitor cells.
Core tip: Previously, we demonstrated that mesenchymal stem cells could be expanded from human endocrine and non-endocrine pancreas cell fractions in vitro. However, we were unable to complete cell differentiation of mesenchymal cell intermediates to functional endocrine cells. In this study we utilized a novel cell culture medium to prevent epithelial cell de-differentiation and mesenchymal cell expansion. After epithelial cell expansion in this medium cells were differentiated via our previously described protocol and we confirmed by lineage tracing, flow cytometry, immunostaining and real-time polymerase chain reaction that islet progenitors reside in the pancreatic epithelium and are not derived via a mesenchymal cell intermediate.