Bruin Jennifer E, Erener Suheda, Vela Javier, Hu Xiaoke, Johnson James D, Kurata Harley T, Lynn Francis C, Piret James M, Asadi Ali, Rezania Alireza, Kieffer Timothy J
Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.
Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
Stem Cell Res. 2014 Jan;12(1):194-208. doi: 10.1016/j.scr.2013.10.003. Epub 2013 Oct 16.
Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day, 7-stage protocol. Cultures were ~90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17, but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine, but not glucose in perifusion studies. Compared to adult human islets, hESC-derived cells expressed ~10-fold higher levels of glucose transporter 1 (GLUT1) mRNA, but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However, GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells, whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays, hESC-derived cells displayed mild potassium channel activity, but no responsiveness to glucose, metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells, suggesting a lack of functional KATP channels at the cell surface. In addition, KATP channel subunit transcript levels were not at a 1:1 ratio, as would be expected (SUR1 levels were ~5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells, and along with lack of GLUT1, may explain the absence of glucose-stimulated insulin secretion.
人类胚胎干细胞（hESCs）被用作人类胰腺发育的模型系统，以研究胎儿胰腺发育过程中出现的多激素细胞的特征。使用一个33天、7个阶段的方案在体外将hESCs分化为类胎儿胰腺细胞。到第11天，培养物中约90 - 95%为PDX1阳性，到第17天，70 - 75%为NKX6.1阳性。多激素细胞在第17天呈散在分布，但到第33天发育成表达关键转录因子的胰岛样簇。在第17天首次检测到人类C肽和胰高血糖素分泌，此后随着INS和GCG转录水平的升高而增加。在灌注研究中，hESC来源的细胞对氯化钾和精氨酸有反应，但对葡萄糖无反应。与成人胰岛相比，hESC来源的细胞表达的葡萄糖转运蛋白1（GLUT1）mRNA水平高约10倍，但葡萄糖激酶（GCK）水平相似。原位杂交证实内分泌细胞内存在GLUT1转录本。然而，GLUT1蛋白不在这群细胞中表达，而是主要在非内分泌细胞中观察到，而GCK在胰岛素阳性细胞中共表达。在铷外流试验中，hESC来源的细胞表现出轻度的钾通道活性，但对葡萄糖、代谢抑制剂或格列本脲无反应。蛋白质印迹实验显示，hESC来源的细胞中不存在较高分子量的SUR1条带，表明细胞表面缺乏功能性的KATP通道。此外，KATP通道亚基转录水平并非预期的1:1比例（SUR1水平比KIR6.2低约5倍）。将不同比例的SUR1:KIR6.2质粒转染到COSM6细胞中，发现铷外流对SUR1的减少特别敏感。这些数据表明，SUR1:KIR6.2比例受损可能导致hESC来源的胰岛内分泌细胞中观察到的KATP通道缺陷，并且与GLUT1的缺乏一起，可能解释了葡萄糖刺激的胰岛素分泌缺失的原因。
