In vitro cultivation of human islets from expanded ductal tissue
In vitro cultivation of human islets from expanded ductal tissue
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DOI：
10.1073/pnas.97.14.7999
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发表时间：
2000-07-05
影响因子：
11.1
通讯作者：
O'Neil, JJ
中科院分区：
文献类型：
--
作者：
Bonner-Weir, S;Taneja, M;O'Neil, JJ
1型和2型糖尿病胰岛移植成功的一个主要障碍是胰岛素产生组织的供应不足。这种对可移植人类胰岛的需求刺激了扩大现有胰岛和/或培育新的胰岛的努力。为了验证成人胰管组织可以在体外扩增和分化形成胰岛细胞的假设，从8个人胰岛分离株中通常丢弃的消化后的胰腺组织在允许导管细胞作为单层扩张的条件下培养，然后在细胞上覆盖一层薄薄的Matrigel。通过这种操作，单层上皮细胞形成了三维的导管囊肿结构，直径50到150微米的胰岛状内分泌细胞簇从这些结构中萌芽。经过3-4周的培养，每瓶的胰岛素含量增加了10-15倍，DNA含量增加了7倍。免疫荧光显示培养的人胰岛芽由细胞角蛋白19阳性的导管细胞和激素阳性的胰岛细胞组成。偶见细胞中胰岛素和非p细胞激素的双重染色，表明未成熟细胞仍处于分化过程中。在培养超过24小时的胰岛素分泌研究中，与5 mM葡萄糖的基础分泌相比，20 mM葡萄糖刺激的包囊/培养的人胰岛芽分泌的胰岛素增加2.3倍。因此，人胰腺导管组织可以在培养中扩增，然后在体外定向分化为葡萄糖反应性胰岛组织。该方法可能为移植提供一种新的胰岛细胞来源。
A major obstacle to successful islet transplantation for both type 1 and 2 diabetes is an inadequate supply of insulin-producing tissue. This need for transplantable human islets has stimulated efforts to expand existing pancreatic islets and/or grow new ones. To test the hypothesis that human adult duct tissue could be expanded and differentiated in vitro to form islet cells, digested pancreatic tissue that is normally discarded from eight human islet isolations was cultured under conditions that allowed expansion of the ductal cells as a monolayer whereupon the cells were overlaid with a thin layer of Matrigel. With this manipulation, the monolayer of epithelial cells formed three-dimensional structures of ductal cysts from which 50- to 150-mu m diameter islet-like clusters of pancreatic endocrine cells budded. Over 3-4 weeks culture the insulin content per flask increased 10- to 15-fold as the DNA content increased up to 7-fold. The cultivated human islet buds were shown by immunofluorescence to consist of cytokeratin 19-positive duct cells and hormone-positive islet cells. Double staining of insulin and non-p cell hormones in occasional cells indicated immature cells still in the process of differentiation. Insulin secretion studies were done over 24 h in culture, Compared with their basal secretion at 5 mM glucose, cysts/cultivated human islet buds exposed to stimulatory 20 mM glucose had a 2.3-fold increase in secreted insulin. Thus, duct tissue from human pancreas can be expanded in culture and then be directed to differentiate into glucose responsive islet tissue in vitro. This approach may provide a potential new source of pancreatic islet cells for transplantation.