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.
World J Diabetes. 2014 Feb 15;5(1):59-68. doi: 10.4239/wjd.v5.i1.59.
To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm that β-cell progenitors reside within the pancreatic epithelium.
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).
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).
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.
在体外最大限度地减少胰腺间充质细胞的扩增，并证实β细胞祖细胞存在于胰腺上皮内。
由于间充质干细胞（MSC）的扩增和过度生长，胰腺上皮内的祖细胞无法在体外进行特征描述，尽管β细胞去分化和通过上皮-间充质转化（EMT）扩增 MSC 中间产物可能会产生β细胞祖细胞。从内分泌和非内分泌组织中扩增和分化胰腺上皮细胞，在一种新型的胰腺上皮细胞扩增培养基中添加已知支持上皮细胞生长的生长因子（地塞米松、表皮生长因子、3,5,3'-三碘甲状腺素、牛脑提取物）。在细胞分化之前，细胞还被单个和双慢病毒报告基因感染。增强型绿色荧光蛋白由大鼠胰岛素 1 启动子控制，单体红色荧光蛋白由小鼠 PDX1 启动子控制。结合慢病毒示踪，在胰腺培养基中扩增和分化的细胞通过流式细胞术（BD 荧光激活细胞分选）、免疫染色和实时聚合酶链反应（PCR）（7900HT Fast Realtime PCR System）进行鉴定。
在 10%血清存在的情况下，MSC 在体外迅速扩增，而上皮细胞群体减少。波形蛋白（+）细胞的百分比从 22%±5.83%增加到 80.43%±3.24%（14 天）和 99.00%±0.0%（21 天），上皮细胞的百分比从 74.71%±8.34%减少到 26.57%±9.75%（14 天）和 4.00%±1.53%（21 天），所有时间点均 P<0.01。我们的新型胰腺上皮细胞扩增培养基保留了上皮细胞表型，并最大限度地减少了上皮细胞去分化和 EMT。与对照组相比，在我们的上皮培养基中扩增的细胞含有明显更少的间充质细胞（波形蛋白（+））（44.87%±4.93%对 95.67%±1.36%；P<0.01）。在细胞分化过程中，慢病毒报告表明，PDX1（+）和胰岛素（+）细胞位于贴壁上皮细胞聚集物中，而不是对照组中的波形蛋白（+）细胞（间充质细胞）。与起始胰岛相比，分化细胞的 PDX1、胰岛素、PAX4 和 RFX 的基因表达至少增加了两倍（P<0.05）。
PDX1（+）细胞局限于贴壁上皮细胞聚集物中，而不是波形蛋白（+）细胞（间充质细胞），这表明 EMT 不是产生胰腺祖细胞的机制。
