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：
8508552
负责人：
I-Ping Chen
金额：
$ 24.9万
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：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8508552
关键词：
Acetone Acid Phosphatase Actins Adhesions Affect Alanine Animal Model BMP4 Biological Assay Biology Bone Diseases CD34 gene Calcitonin Receptor Cell Line Cell Nucleus Cells Childhood Cholecalciferol Chromosomes Citrates Confocal Microscopy Culture Media Cytoskeleton Defect Development Disease Dissociation Dysplasia Eating Ectoderm Endoderm Energy Transfer Ensure Exploratory/Developmental Grant for Diagnostic Cancer Imaging Family member Fibroblasts Film Gene Expression Generations Genes Genetic Genomics Germ Layers Gifts Goals Guanosine Triphosphate Phosphohydrolases Hematopoietic Hematoxylin and Eosin Staining Method Hip region structure Histologic Human Image Immunoblotting Immunofluorescence Immunologic Individual Interleukin 2 Receptor Interleukin-3 Karyotype determination procedure Knock-in Mouse Knockout Mice Laboratories Leukocytes Life MMP9 gene Macrophage Colony-Stimulating Factor Mentors Methods Minerals Molecular Profiling Morphology Movement Mus Mutation Operative Surgical Procedures Osteoclasts PTPRC gene Pathogenesis Patients Peripheral Blood Mononuclear Cell Phalloidine Phase Phenylalanine Plague Publishing Quality Control RNA Rare Diseases Regulation Reporting Research Resistance Reverse Transcriptase Polymerase Chain Reaction Rhodamine Role Sampling Science Sendai virus Serine Services Shapes Skin Slice Slide Solutions Staining method Stains Stem cells Stromal Cells System TNFSF11 gene Teratoma Testing Time Tissues Tyrosine Phosphorylation Undifferentiated Work abstracting base biocoating bone bone cell c-myc Genes calcium phosphate cathepsin K cell type collagenase craniofacial deciduous tooth disease mechanisms study established cell line human stem cells improved in vivo induced pluripotent stem cell long bone magnetic beads matrigel migration monolayer mouse model peripheral blood response retroviral transduction rho skeletal disorder stage-specific embryonic antigen 4 stem stem cell biology vector volunteer

项目摘要

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.

项目概要/摘要已发现 300 多种罕见的遗传性骨病，但这些疾病的治疗通常很有限，因为对其发病机制知之甚少。原代细胞/组织的缺乏和缺乏合适的动物模型一直困扰着罕见疾病的研究。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途径。在此应用中，我计划使用患者特异性 IPS 细胞来研究颅骨干骺端发育不良 (CMD) 的疾病机制，重点关注破骨细胞 (OC)（骨吸收细胞）的作用。 CMD 始于儿童时期，伴有颅面骨增厚和长骨形状异常。它的终生进展会导致一些患者危及生命。迄今为止，除了重复手术之外，没有其他治疗方法。先前对携带导致 CMD 的 Ank 突变的敲入 (K1) 小鼠模型的研究揭示了 Ank[Ki/Ki] 小鼠的 OC 缺陷。在 CMD 患者的人外周血培养中也发现了类似的结果。 Ank[Ki/Ki] OCs 也表现出运动较慢且肌动蛋白组织异常。提出了两个具体目标来检验以下假设：CMD 引起的 ANK 突变通过对肌动蛋白细胞骨架产生负面影响来降低个体破骨细胞活性。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 OC，以确定 OC 形成、基质和矿物质吸收、分别通过粘附测定和活细胞延时成像观察 OC 标记基因、粘附和迁移。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态。肌动蛋白生物学中的两个关键调节机制，即 GTPase 家族成员 Rac、Rho 和 Cdc42 的激活，以及对照和 CMD 患者 iPS 衍生 OC 中的酪氨酸磷酸化，将通过活性 GTPase Pull-down 测定、免疫染色和免疫印迹进行检查。或者，我们将使用基于 FRET（荧光共振能量转移）的分析来确定 CMD 患者和对照的破骨细胞中 Rac、Rho 和 Cdc42 的动态调节。本研究的长期目标是将 CMD 建立为研究罕见遗传性骨骼疾病机制的范例。