Original Article
Copyright ©2014 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Diabetes. Feb 15, 2014; 5(1): 59-68
Published online Feb 15, 2014. doi: 10.4239/wjd.v5.i1.59
Identification and differentiation of PDX1 β-cell progenitors within the human pancreatic epithelium
Karen L Seeberger, Sarah J Anderson, Cara E Ellis, Telford Y Yeung, Gregory S Korbutt
Karen L Seeberger, Sarah J Anderson, Cara E Ellis, Telford Y Yeung, Gregory S Korbutt, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada
Gregory S Korbutt, Department of Surgery, University of Alberta, Edmonton, Alberta T6G 2E1, Canada
Gregory S Korbutt, Alberta Diabetes Institute, 5-002 Li Ka Shing Center, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada
Author contributions: Seeberger KL and Anderson SJ contributed equally to this work; Seeberger KL and Korbutt GS designed the research; Seeberger KL and Anderson SJ performed the research; Ellis CE and Yeung TY contributed new reagents/analytic tools; Seeberger KL, Anderson SJ, Ellis CE and Yeung TY analyzed the data; and Seeberger KL wrote the paper.
Supported by Canadian Institutes of Health Research, No. MOP8030; and the Alberta Diabetes Institute
Correspondence to: Gregory S Korbutt, Professor, Alberta Diabetes Institute, 5-002 Li Ka Shing Center, University of Alberta, Edmonton, 116 St. and 85 Ave, Alberta, T6G 2E1, Canada. korbutt@ualberta.ca
Telephone: +1-780-4924657 Fax: +1-780-4925501
Received: September 13, 2013
Revised: November 27, 2013
Accepted: December 13, 2013
Published online: February 15, 2014
Processing time: 159 Days and 9.1 Hours
Abstract

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.

Keywords: Differentiation; Epithelial; Epithelial-mesenchymal transition; Mesenchymal; PDX1; Insulin; Progenitor; Vimentin

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.