β-Cell Differentiation of Human Pancreatic Duct-Derived Cells After In Vitro Expansion
β-Cell Differentiation of Human Pancreatic Duct-Derived Cells After In Vitro Expansion
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DOI：
10.1089/cell.2014.0025
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发表时间：
2014-12-01
影响因子：
1.6
通讯作者：
Lysy, Philippe A.
中科院分区：
文献类型：
--
作者：
Corritore, Elisa;Dugnani, Erica;Lysy, Philippe A.
β细胞替代疗法是一个很有前景的研究领域，目前正在评估用于临床的新细胞来源。胰腺上皮细胞是β细胞工程的有力候选者，但其大规模扩张尚未得到证实。在这里，我们描述了纯化的人胰管细胞 (DC) 的有效扩增和 β 细胞分化。当在内皮生长促进培养基中培养时，纯化的 CA19-9(+) 细胞广泛增殖，并在 9 代中实现了多达 22 次群体倍增。在增殖时，人胰管源性细胞 (HDDC) 下调大多数 DC 标记物，但它们保留了较低的 CK19 和 SOX9 基因表达。 HDDC 获得了间充质特征，但与成纤维细胞或胰腺基质细胞不同。导管和间质标记物的共表达表明 HDDC 是通过部分上皮间质转化 (EMT) 衍生自 DC。与 EMT 抑制剂 A83-01 孵育后，CA19-9(+) 细胞培养物中 HDDC 的出现被阻断，这也支持了这一点。经过模拟胰腺发育的分化方案后，HDDC 群体含有约 2% 的未成熟胰岛素生成细胞，并表现出对葡萄糖无反应的胰岛素分泌。间充质表型的下调改善了分化 HDDC 的 β 细胞基因表达谱，而不影响胰岛素蛋白的表达和分泌。我们表明，胰管代表了工程大量具有治疗糖尿病潜力的β样细胞的新来源。
beta-Cell replacement therapy is a promising field of research that is currently evaluating new sources of cells for clinical use. Pancreatic epithelial cells are potent candidates for beta-cell engineering, but their large-scale expansion has not been evidenced yet. Here we describe the efficient expansion and beta-cell differentiation of purified human pancreatic duct cells (DCs). When cultured in endothelial growth-promoting media, purified CA19-9(+) cells proliferated extensively and achieved up to 22 population doublings over nine passages. While proliferating, human pancreatic duct-derived cells (HDDCs) downregulated most DC markers, but they retained low CK19 and SOX9 gene expression. HDDCs acquired mesenchymal features but differed from fibroblasts or pancreatic stromal cells. Coexpression of duct and mesenchymal markers suggested that HDDCs were derived from DCs via a partial epithelial-to-mesenchymal transition (EMT). This was supported by the blockade of HDDC appearance in CA19-9(+) cell cultures after incubation with the EMT inhibitor A83-01. After a differentiation protocol mimicking pancreatic development, HDDC populations contained about 2% of immature insulin-producing cells and showed glucose-unresponsive insulin secretion. Downregulation of the mesenchymal phenotype improved beta-cell gene expression profile of differentiated HDDCs without affecting insulin protein expression and secretion. We show that pancreatic ducts represent a new source for engineering large amounts of beta-like-cells with potential for treating diabetes.