Induced Pluripotent Stem Cell Reprogramming by Integration-Free Sendai Virus Vectors from Peripheral Blood of Patients with Craniometaphyseal Dysplasia
Induced Pluripotent Stem Cell Reprogramming by Integration-Free Sendai Virus Vectors from Peripheral Blood of Patients with Craniometaphyseal Dysplasia
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DOI：
10.1089/cell.2013.0037
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发表时间：
2013-12-01
影响因子：
1.6
通讯作者：
Reichenberger, Ernst J.
中科院分区：
文献类型：
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作者：
Chen, I-Ping;Fukuda, Keiichi;Reichenberger, Ernst J.
罕见遗传性骨病的研究往往是有限的，由于组织标本的不可用性和缺乏动物模型完全复制表型特征。颅后骺发育不良是一种罕见的单基因遗传性疾病，其特征是颅面骨的骨质增生，同时伴有长骨的畸形。常染色体显性CMD突变已在ANK基因（ANKH）中鉴定。在这里，我们描述了一种简单而有效的方法，从8名CMD患者和5名健康对照的外周血培养的贴壁细胞重编程为人诱导多能干细胞（hiPSC）。通过Ficoll梯度从5- 7 mL全血中分离外周血单核细胞（PBMC），在细胞因子存在下扩增，并用编码OCT 3/4、SOX 2、KLF 4和c-MYC的仙台病毒（SeV）载体转导。SeV载体是一种细胞质RNA载体，在增殖10-13代后从宿主细胞中丢失。这些hiPSC表达干细胞标志物，具有正常的核型，并且能够在体外形成胚状体以及在体内形成畸胎瘤。将这些患者特异性iPSC进一步分化为成骨细胞和破骨细胞可以为研究CMD突变对骨骼的影响提供有用的工具，这种方法可以应用于其他罕见遗传性肌肉骨骼疾病的疾病建模。
Studies of rare genetic bone disorders are often limited due to unavailability of tissue specimens and the lack of animal models fully replicating phenotypic features. Craniometaphyseal dysplasia (CMD) is a rare monogenic disorder characterized by hyperostosis of craniofacial bones concurrent with abnormal shape of long bones. Mutations for autosomal dominant CMD have been identified in the ANK gene (ANKH). Here we describe a simple and efficient method to reprogram adherent cells cultured from peripheral blood to human induced pluripotent stem cells (hiPSCs) from eight CMD patients and five healthy controls. Peripheral blood mononuclear cells (PBMCs) were separated from 5-7mL of whole blood by Ficoll gradient, expanded in the presence of cytokines and transduced with Sendai virus (SeV) vectors encoding OCT3/4, SOX2, KLF4, and c-MYC. SeV vector, a cytoplasmic RNA vector, is lost from host cells after propagation for 10-13 passages. These hiPSCs express stem cell markers, have normal karyotypes, and are capable of forming embryoid bodies in vitro as well as teratomas in vivo. Further differentiation of these patient-specific iPSCs into osteoblasts and osteoclasts can provide a useful tool to study the effects CMD mutations on bone, and this approach can be applied for disease modeling of other rare genetic musculoskeletal disorders.