The use of patient-specific iPS cells to identify osteoclast defects in CMD

项目概要/摘要 目前已经发现了300多种罕见的遗传性骨病，但对这些疾病的治疗通常是有限的，因为对它们的发病机制知之甚少。罕见病的研究一直受到原代细胞/组织不可用和缺乏合适动物模型的困扰。患者特异性诱导多能干（iPS）细胞生物学的最新进展为研究患者骨细胞开辟了新的途径。在本申请中，我计划使用患者特异性IPS细胞来研究颅骺发育不良（CMD）的疾病机制，重点是破骨细胞（OC），骨吸收细胞的作用。CMD的发病始于儿童时期，伴随颅面骨增厚和长骨形状异常。其终身进展导致一些患者危及生命的后果。到目前为止，除了重复手术外，没有其他治疗方法。先前在携带引起CMD的Ank突变的敲入（KI）小鼠模型中的研究揭示了Ank[Ki/Ki]小鼠中的OC缺陷。在CMD患者的人外周血培养中发现了类似的结果。Ank[Ki/Ki] OC也表现出较慢的运动与异常肌动蛋白组织。提出了两个具体的目标来检验这一假设，CMD引起的ANK突变，减少个别破骨细胞活性的负面影响肌动蛋白细胞骨架。在目标1中，申请人将比较来自健康对照和CMD患者的iPS衍生的OC，以鉴定OC形成、基质和矿物质吸收、 OC标记基因，粘附和迁移的粘附试验和活细胞延时成像。 在目标2中，申请人将通过共聚焦显微镜研究CMD和对照OC中肌动蛋白细胞骨架的组织和动力学。肌动蛋白生物学中的两个关键调节机制，GTdR家族成员Rac、Rho和Cdc 42的激活，以及对照和CMD患者的iPS衍生的OC中的酪氨酸磷酸化，将通过活性GTdR下拉测定、免疫染色和免疫印迹来检查。可选择地， 我们将使用基于FRET（荧光共振能量转移）的分析来确定来自CMD患者和对照的活体破骨细胞中Rac、Rho和Cdc 42的动态调节。本研究的长期目标是建立CMD作为研究罕见遗传性骨骼疾病机制的范例。

Project Summary/Abstract More than 300 rare genetic bone diseases have been identified but treatment for these disorders is usually limited because little of their pathogeneses is known. Studies of rare diseases have been plagued by the unavailability of primary cells/tissues and lack of suitable animal models. Recent advance in patient-specific induced pluripotent stem (iPS) cell biology opened new avenues for studying bone cells from patients. In this application, i plan to use patient-specific IPS cells to study disease mechanisms of craniometaphyseal dysplasia (CMD) with a focus on the role of osteoclasts (OCs), the bone resorbing cells. The onset of CMD begins in childhood with thickening of craniofacial bones and abnormally shaped long bones. Its lifelong progression leads to life-threatening consequences in some patients. To date, there is no treatment other than repetitive surgery. Previous studies in a knock-in (Kl) mouse model carrying a CMD-causing Ank mutation revealed OC defects in Ank[Ki/Ki] mice. Similar results were found in human peripheral blood cultures of CMD patients. Ank[Ki/Ki] OCs also showed slower movement with abnormal actin organization. Two specific aims are proposed to test the hypothesis that CMD-causing ANK mutations reduce individual osteoclast activity by negatively affecting the actin cytoskeleton. In Aim 1 the applicant will compare iPS-derived OCs from healthy controls and CMD patients to identify differences in OC formation, matrix and mineral resorption, expression of OC marker genes, adhesion and migration by adhesion assays and live-cell time-lapse imaging, respectively. In Aim 2 the applicant will study the organization and dynamics of the actin cytoskeleton in CMD and control OCs by confocal microscopy. Two critical regulatory mechanisms in actin biology, the activation of GTPase family members Rac, Rho and Cdc42, and tyrosine phosphorylation in iPS-derived OCs of controls and CMD patients will be examined by active GTPase pull-down assays, immunostaining and immunoblots. Alternatively, we will use a FRET (fluorescent resonance energy transfer)-based analysis to determine the dynamic regulation of Rac, Rho and Cdc42 in life osteoclasts from CMD patients and controls. The long-term goal of this study is to establish CMD as a paradigm for studying mechanisms of rare genetic skeletal disease.