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

描述(申请人提供)：300多种罕见的遗传性骨骼疾病已被确认，但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。目前研究这些疾病的方法包括动物模型或从患者的血液或骨髓中产生骨细胞。然而，反复从患者身上收集样本往往是不切实际的，可能是不道德的，而动物模型往往无法复制这些疾病的完整特征。患者特异性诱导多能干细胞生物学的最新进展为研究患者的骨细胞开辟了新的途径。在这份为期5年的计划中，申请者计划使用患者特有的iPS细胞来研究颅骨干骺端发育不良(CMD)，重点是破骨细胞(OCS)，即骨吸收细胞。CMD起病于儿童时期，主要表现为头面部骨骼增厚和长骨形态异常。它的终生进展会导致一些患者出现危及生命的后果。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CMD引起的Ank突变的敲入(Ki)小鼠模型中的研究发现，AnkKI/Ki小鼠存在OC缺陷。在CMD患者的人外周血培养中也发现了类似的结果。AnkKI/KI OCS运动减慢，肌动蛋白组织异常。为了验证从CMD小鼠模型中产生的假设，即CMD引起的ANK突变通过负面影响肌动蛋白细胞骨架而降低个体破骨细胞的活性，提出了四个具体目标。在目标1和2(K99阶段)中，申请者将优化1)从对照组和CMD患者的成纤维细胞或棚(来自人类脱落乳牙的干细胞)中培养iPS细胞的方法，以及2)将iPS细胞分化为OCs的方法。在目标3(R00阶段)中，申请者将比较来自健康对照组和CMD患者的iPS来源的OCs，以确定OC的形成、功能、OC标记基因的表达、通过TRAP染色、吸收坑分析、实时聚合酶链式反应、黏附分析和活细胞时间推移成像分别进行的黏附和迁移的差异。在目标4(R00阶段)，申请人将通过共聚焦显微镜研究肌动蛋白细胞骨架的组织和动力学，以及ANK和肌动蛋白在对照组和CMD OCS中的共存情况。肌动蛋白生物学中的两个关键调控机制，即GTPase家族成员Rac、Rho和CDc42的激活，以及对照组和CMD患者iPS来源的OCS中的酪氨酸磷酸化，将通过活性GTP酶下拉试验、免疫染色和免疫印迹来检测。这项研究的最终目标是使用CMD作为范例，建立研究罕见遗传性骨骼疾病的新工具。干细胞和骨生物学是UCHC牙医学院的主要兴趣领域。研究环境非常适合执行这一项目，并在牙医/科学家轨道上发展申请人的职业生涯。 公共卫生相关性：这项应用使用人类诱导多能干细胞(IPS)来研究破骨细胞缺陷在颅骨干骺端发育不良(CMD)中的缺陷，这是一种罕见的遗传性骨骼疾病。这项研究有可能为CMD的治疗提供新的治疗靶点，并提高我们对破骨细胞生物学的理解，这可能有助于其他罕见的遗传性骨骼疾病和一些常见的破骨细胞相关疾病的研究，如骨质疏松或骨化症。

DESCRIPTION (provided by applicant): 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. Current methods for studying these disorders include animal models or generating bone cells from patient blood or bone marrow. However, collecting specimens repeatedly from patients is often impractical and may be unethical, while animal models often fail to replicate the complete features of those diseases. Recent advance in patient-specific induced pluripotent stem (iPS) cell biology opened new avenues for studying bone cells from patients. In this 5-year proposal, the applicant plans to use patient-specific iPS cells to study craniometaphyseal dysplasia (CMD) with a focus on osteoclasts (OCs), the bone resorbing cells. The onset of CMD begins in childhood with thickening of craniofacial bones and abnormal shape of 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 (KI) mouse model carrying a CMD-causing Ank mutation revealed OC defects in AnkKI/KI mice. Similar results were found in human peripheral blood cultures of CMD patients. AnkKI/KI OCs also showed slower movement with abnormal actin organization. To test the hypothesis generated from the CMD mouse model that CMD-causing ANK mutations reduce individual osteoclast activity by negatively affecting the actin cytoskeleton, four specific aims are proposed. In Aims 1 and 2 (K99 phase), the applicant will optimize the methods 1) for generating iPS cells from fibroblasts or SHEDs (stem cells from human exfoliated deciduous teeth) of control individuals and CMD patients and 2) for differentiating iPS cells into OCs. In Aim 3 (R00 phase), the applicant will compare iPS- derived OCs from healthy controls and CMD patients to identify differences in OC formation, function, expression of OC marker genes, adhesion and migration by TRAP staining, resorption pit assays, real-time PCR, adhesion assays and live-cell time-lapse imaging, respectively. In Aim 4 (R00 phase), the applicant will study the organization and dynamics of the actin cytoskeleton as well as the colocalization of ANK and actin in control and CMD 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. The ultimate goal of this study is to use CMD as paradigm to establish novel tools for studying rare genetic skeletal disorders. Stem cell and bone biology are areas of major interest at School of Dental Medicine at UCHC. The research environment is exquisitely suited to perform this project and to develop the applicant's career in the Dentist/Scientist track. PUBLIC HEALTH RELEVANCE: This application uses human induced pluripotent stem (iPS) cells to study osteoclast defects in craniometaphyseal dysplasia (CMD), a rare genetic bone disorder. This study has the potential to reveal novel therapeutic targets for CMD treatment and to improve our understanding of osteoclast biology, which may benefit research in other rare genetic skeletal disorders and some common osteoclast-related disorders, such as osteoporosis or osteopetrosis.