Impaired adipogenic capacity in induced pluripotent stem cells from lipodystrophic patients with BSCL2 mutations
Impaired adipogenic capacity in induced pluripotent stem cells from lipodystrophic patients with BSCL2 mutations
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DOI：
10.1016/j.metabol.2015.12.015
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发表时间：
2016-04-01
影响因子：
9.8
通讯作者：
Nakao, Kazuwa
中科院分区：
文献类型：
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作者：
Mori, Eisaku;Fujikura, Junji;Nakao, Kazuwa
Objective.先天性全身性脂肪营养不良（Congenital Generalized Lipodystrophy，CGL）是一种常染色体隐性遗传疾病，其特征为脂肪组织明显缺乏、极端胰岛素抵抗、高甘油三酯血症、肝脂肪变性和早发性糖尿病。BSCL 2/SEIPIN基因的突变导致最严重形式的CGL。本研究的目的是从携带BSCL 2/SEIPIN突变的CGL患者中产生诱导多能干细胞（iPS）。皮肤活组织检查获自两名在BSCL 2/SEIPIN中具有不同无义突变（E189 X和R275 X）的CGL日本患者。用编码OCT 4、SOX 2、c-MYC和KLF 4的逆转录病毒感染由此获得的成纤维细胞。通过检查多能性标志物（碱性磷酸酶、SSEA-4、TRA-1-60和NANOG）的表达以及它们通过形成拟胚体在体外分化为三个胚层和在体内形成畸胎瘤的能力来评价所产生的iPS细胞的多能性。油红0和脂肪分化相关蛋白（ADRP）染色检测BSCL 2-iPS细胞的成脂能力。还使用野生型BSCL 2的稳定表达进行拯救实验。采用免疫共沉淀法研究SEIPIN与ADRP的相互作用。iPS细胞由两名CGL患者的成纤维细胞产生。每个患者来源的iPS（BSCL 2-iPS）克隆都显示出多能性的所有特征，并且可以通过形成拟胚体在体外分化成所有三个胚层的衍生物，并且在注射到小鼠睾丸中后形成畸胎瘤。BSCL 2-iPS细胞保持BSCL 2中的突变并且缺乏完整的BSCL 2。在成脂分化后，与来自健康个体的iPS细胞相比，BSCL 2-iPS细胞表现出脂滴形成的显著减少，伴随着ADRP的弥漫性胞质分布。在BSCL 2-iPS细胞中，BSCL 2的强制表达不仅挽救了脂质积累缺陷，而且恢复了ADRP的胞浆点状定位。免疫共沉淀结果显示SEIPIN与ADR有相互作用。BSCL 2-iPS细胞在体外重现了脂肪营养不良的表型，可以提供有价值的模型，用于研究脂质积累的生理学和人类脂肪营养不良的病理学。我们发现BSCL 2定义了ADRP的定位，ADRP在脂质积累和脂肪形成分化中起作用。(C)2016 Elsevier Inc. All rights reserved.
Objective. Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder characterized by marked scarcity of adipose tissue, extreme insulin resistance, hypertriglyceridemia, hepatic steatosis and early-onset diabetes. Mutation of the BSCL2/SEIPIN gene causes the most severe form of CGL. The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with CGL harboring BSCL2/SEIPIN mutations.Methods. Skin biopsies were obtained from two Japanese patients with CGL harboring different nonsense mutations (E189X and R275X) in BSCL2/SEIPIN. The fibroblasts thus obtained were infected with retroviruses encoding OCT4, SOX2, c-MYC, and KLF4. The generated iPS cells were evaluated for pluripotency by examining the expression of pluripotency markers (alkaline phosphatase, SSEA-4, TRA-1-60, and NANOG) and their ability to differentiate to three germ layers in vitro by forming embryoid bodies, and to form teratomas in vivo. Adipogenic capacity of differentiated BSCL2-iPS cells was determined by oil red 0 and adipose differentiation-related protein (ADRP) staining. Rescue experiments were also performed using stable expression of wild-type BSCL2. A coimmunoprecipitation assay was conducted to investigate the interaction of SEIPIN with ADRP.Results. iPS cells were generated from fibroblasts of the two patients with CGL. Each of the patient-derived iPS (BSCL2-iPS) clones showed all of the hallmarks of pluripotency and could differentiate into derivatives of all three germ layers in vitro by forming embryoid bodies, and form teratomas after injection into mouse testes. BSCL2-iPS cells maintained the mutations in BSCL2 and lacked intact BSCL2. Upon adipogenic differentiation, BSCL2-iPS cells exhibited marked reduction of lipid droplet formation concomitant with diffuse cytoplasmic distribution of ADRP, compared with iPS cells from healthy individuals. Forced expression of BSCL2 not only rescued the lipid accumulation defects, but also restored cytoplasmic punctate localization of ADRP in BSCL2-iPS cells. Coimmunoprecipitation indicated SEIPIN interacted with ADRP.Conclusion. BSCL2-iPS cells that recapitulate the lipodystrophic phenotypes in vitro could provide valuable models with which to study the physiology of lipid accumulation and the pathology of human lipodystrophy. We found that BSCL2 defines the localization of ADRP, which has a role in lipid accumulation and adipogenic differentiation. (C) 2016 Elsevier Inc. All rights reserved.