Background

Moyamoya disease (MMD) is an occlusive cerebrovascular disease, causing stroke in children and young adults with unknown etiology. The fundamental pathology is fibrocellular intimal thickening of cerebral arteries, in which vascular smooth muscle cells (VSMCs) are observed as one of the major cell types. Although the characteristics of circulating smooth muscle progenitor cells have been previously reported, the VSMCs are poorly characterized in MMD. We aimed to characterize VSMCs in MMD using induced pluripotent stem cell (iPSC)-technology.

Methods

We differentiated VSMCs from neural crest stem cells (NCSCs) using peripheral blood mononuclear cell-derived iPSCs and compared biological and transcriptome features under naïve culture conditions between three independent healthy control (HC) subjects and three MMD patients. VSMC transcriptome profiles were also compared to those of endothelial cells (ECs) differentiated from the same iPSCs.

Results

Homogeneous spindle-shaped cells differentiated from iPSCs exhibited smooth muscle cell marker expressions, including α-smooth muscle actin (αSMA, 82.3 ± 6.7% and 81.0 ± 6.7%); calponin (91.3 ± 2.1% and 90.9 ± 1.3%); myosin heavy chain-11 (MYH11, 96.9 ± 0.7% and 97.1 ± 0.3%) without significance of differences between the two groups. Real-time PCR showed few PECAM1 and CD34 gene expressions in both groups, indicating features of differentiated VSMCs. There were no significant differences in cellular proliferation (p = 0.45), migration (p = 0.60), and contractile abilities (p = 0.96) between the two groups. Transcriptome analysis demonstrated similar gene expression profiles of VSMCs in HC subjects and MMD patients with six differentially expressed genes (DEGs); while ECs showed a distinct transcriptome profile in MMD patients with 120 DEGs. The Wnt-signaling pathway was a significant pathway in VSMCs.

Conclusions

This is the first study that established VSMCs from NCSCs using MMD patient-derived iPSCs and demonstrated similar biological function and transcriptome profile of iPSC-derived VMSCs in MMD patients and HC subjects under naïve single culture condition. Comparative transcriptome features between iPSC-derived VSMCs and ECs, displaying distinct transcriptome in the ECs, suggested that pathological traits can be driven by naïve ECs predominantly and VSMCs may require specific environmental factors in MMD, which provides novel insight into the pathophysiology of MMD. Our iPSC derived VSMC model can contribute to further investigations of diagnostic and therapeutic target of MMD in addition to the current iPSC derived EC model.

中文翻译：

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烟雾病IPS细胞衍生的血管平滑肌细胞-与内皮细胞转录组的比较特征。

背景

Moyamoya病（MMD）是一种闭塞性脑血管疾病，在病因不明的儿童和年轻人中引起中风。基本病理是脑动脉的纤维细胞内膜增厚，其中血管平滑肌细胞（VSMC）被视为主要细胞类型之一。尽管先前已经报道了循环平滑肌祖细胞的特征，但是VSMC在MMD中的特征很差。我们旨在使用诱导性多能干细胞（iPSC）技术表征MMD中的VSMC。

方法

我们使用外周血单核细胞衍生的iPSC区分了VSMC和神经c干细胞（NCSC），并比较了三名独立健康对照（HC）受试者和三名MMD患者在单纯培养条件下的生物学和转录组特征。还比较了VSMC转录组概况与从相同iPSC分化出的内皮细胞（EC）的概况。

结果

从iPSC分化出来的同质纺锤状细胞表现出平滑肌细胞标志物表达，包括α-平滑肌肌动蛋白（αSMA，82.3±6.7％和81.0±6.7％）；钙蛋白（91.3±2.1％和90.9±1.3％）; 肌球蛋白重链11（MYH11，96.9±0.7％和97.1±0.3％），两组之间无差异。实时PCR显示两组中几乎没有PECAM1和CD34基因表达，表明分化的VSMC的特征。细胞增殖（p  = 0.45），迁移（p  = 0.60）和收缩能力（p = 0.96）。转录组分析表明，在具有六种差异表达基因（DEG）的HC受试者和MMD患者中，VSMC的基因表达谱相似。而ECs在120DEG的MMD患者中表现出独特的转录组特征。Wnt信号通路是VSMC中的重要途径。

结论

这是第一项使用MMD患者来源的iPSC从NCSC建立VSMC的研究，并证明了在单纯的单一培养条件下，MMD患者和HC受试者中iPSC来源的VMSC的生物学功能和转录组谱具有相似的生物学功能。iPSC衍生的VSMC与EC之间的转录组比较特征表明EC中表现出独特的转录组，这表明病理特征可能主要由幼稚的EC驱动，而VSMC可能需要MMD中的特定环境因素，这为MMD的病理生理学提供了新颖的见解。除了当前的iPSC衍生的EC模型之外，我们的iPSC衍生的VSMC模型还可以为MMD的诊断和治疗目标的进一步研究做出贡献。