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

使用患者特异性 iPS 细胞识别 CMD 中的破骨细胞缺陷

基本信息

批准号：
8143488
负责人：
I-Ping Chen
金额：
$ 12.1万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-15 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8143488
关键词：
Actins Adhesions Affect Albers-Schonberg disease Animal Model Area Biological Assay Biology Blood Bone Diseases Bone Marrow Cells Characteristics Childhood Confocal Microscopy Cytoskeleton Data Defect Dental Pulp Dental Schools Dentists Disease Dysplasia Environment Family member Fibroblasts Flare Gender Gene Expression Genes Genetic Goals Guanosine Triphosphate Phosphohydrolases Human Human Genetics Hyperostosis Image Image Analysis Immigration Immunoblotting Individual Investigation Knock-in Mouse Life Medicine Mentors Methods Modification Molecular Movement Mutation Operative Surgical Procedures Orphan Disease Osteoclasts Osteoporosis Pathogenesis Patients Phase Plague Rare Diseases Regulation Research Reverse Transcriptase Polymerase Chain Reaction Scientist Shapes Skin Specimen Staining method Stains Stem cells System Techniques Testing Time Tissues Training Tyrosine Phosphorylation base bone bone cell career cell motility cellular imaging craniofacial deciduous tooth effective therapy human stem cells improved induced pluripotent stem cell interest long bone migration mouse model new therapeutic target novel peripheral blood pluripotency public health relevance research study rho sex skeletal disorder stem stem cell biology tool

项目摘要

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.

描述（由申请人提供）：已经确定了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。目前研究这些疾病的方法包括动物模型或从患者血液或骨髓中产生骨细胞。然而，从患者身上重复收集标本往往不切实际，而且可能不道德，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（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。在这项为期5年的提案中，申请人计划使用患者特异性iPS细胞来研究颅骨骺发育不良（CMD），重点是破骨细胞（OC），即骨吸收细胞。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。先前在携带导致CMD的Ank突变的基因敲入（KI）小鼠模型中的研究揭示了AnkKI/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。AnkKI/KI OC也显示出较慢的运动与异常肌动蛋白组织。为了检验从CMD小鼠模型产生的假设，即CMD引起的ANK突变通过负面影响肌动蛋白细胞骨架来降低个体破骨细胞活性，提出了四个具体的目标。在目标1和2（K99期）中，申请人将优化方法1）用于从对照个体和CMD患者的成纤维细胞或SHED（来自人脱落乳牙的干细胞）生成iPS细胞，以及2）用于将iPS细胞分化为OC。在目标3（R 00阶段）中，申请方将比较来自健康对照和CMD患者的iPS衍生OC，以分别通过TRAP染色、再吸收陷窝试验、实时PCR、粘附试验和活细胞延时成像确定OC形成、功能、OC标志物基因表达、粘附和迁移的差异。在目标4（R 00阶段）中，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和肌动蛋白在对照和CMD OC中的共定位。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。本研究的最终目的是以CMD为范式，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牙科医学院的主要兴趣领域。研究环境非常适合执行这个项目，并发展申请人在牙医/科学家轨道的职业生涯。公共卫生关系：本申请使用人诱导多能干细胞（iPS）研究颅骺发育不良（CMD）（一种罕见的遗传性骨疾病）中的破骨细胞缺陷。这项研究有可能揭示CMD治疗的新治疗靶点，并提高我们对破骨细胞生物学的理解，这可能有利于其他罕见遗传性骨骼疾病和一些常见的破骨细胞相关疾病，如骨质疏松症或骨硬化症的研究。