Probing sporadic and familial Alzheimer?s disease using induced pluripotent stem cells.

Our understanding of Alzheimer’s disease (AD) pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of AD. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). We reprogrammed primary fibroblasts from two patients with familial AD (both caused by a duplication of APP1, APPDp), two with sporadic AD (sAD1, 2) and two non-demented control individuals (NDCs) into iPSC lines. Neurons from differentiated cultures were FACS-purified and characterized. Purified cultures contained >90% neurons, clustered with fetal brain mRNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APPDp patients and patient sAD2 exhibited significantly higher levels of secreted Aβ1-40, phospho-tauThr231 (pTau) and active GSK3β (aGSK3β). Neurons from APPDp and sAD2 patients also accumulated large Rab5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not g-secretase inhibitors, caused significant reductions in pTau and aGSK3β levels. These results suggest a direct relationship between APP proteolytic processing, but not Aβ, in GSK3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial AD samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to AD, even though it can take decades for overt disease to manifest in patients. Total RNA extracted from normal hIPSCs, Alzheimer's patient derived hIPSCs, neurons differentiated from hIPSCs, fetal brain, fetal heart, fetal liver and fetal lung

目前我们对阿尔茨海默病（Alzheimer’s disease, AD）发病机制的理解受限于两大瓶颈：一是难以从患者体内获取活神经元，二是无法构建散发性AD（sporadic AD, sAD）的体外模型。通过将患者原代细胞重编程为诱导多能干细胞（induced pluripotent stem cells, iPSCs），或可攻克这些限制。
我们将两名家族性AD患者（均由APP1基因拷贝数重复导致，即APPDp）、两名散发性AD患者（sAD1、sAD2）以及两名非痴呆对照个体（non-demented control individuals, NDCs）的原代成纤维细胞重编程为iPSC细胞系。对分化培养得到的神经元进行荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）纯化并鉴定：纯化后的神经元纯度超过90%，通过基因芯片（microarray）分析可与胎儿脑组织转录本聚类，并能形成功能性突触连接；几乎所有细胞均表现出正常的电生理活性。
与对照组相比，两名APPDp患者以及sAD2患者的iPSC来源纯化神经元，其分泌型Aβ1-40（secreted Aβ1-40）、磷酸化tau蛋白Thr231位点（phospho-tauThr231, pTau）以及活化型糖原合成酶激酶3β（active GSK3β, aGSK3β）的表达水平显著升高。相较于对照组，APPDp与sAD2患者的神经元还会蓄积大量Rab5阳性早期内体（Rab5-positive early endosomes）。使用β分泌酶抑制剂（β-secretase inhibitors）而非γ分泌酶抑制剂（γ-secretase inhibitors）处理纯化神经元，可显著降低pTau与aGSK3β的水平。
上述结果表明，在人类神经元中，APP的蛋白水解加工过程（而非Aβ本身）与GSK3β激活及tau磷酸化存在直接关联。此外我们观察到，携带一名散发性AD患者基因组的神经元，表现出了家族性AD样本中的相关表型。总体而言，本研究证实诱导多能干细胞技术可用于观测与AD相关的病理表型——尽管患者通常需要数十年才会出现显性临床症状。
本研究提取的总RNA样本来源包括：正常人类诱导多能干细胞、阿尔茨海默病患者来源的人类诱导多能干细胞、人类诱导多能干细胞分化得到的神经元、胎儿脑组织、胎儿心脏组织、胎儿肝脏组织以及胎儿肺组织。