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：
8721393
负责人：
I-Ping Chen
金额：
$ 23.1万
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：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8721393
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 Profiling 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.

项目摘要/摘要已经确定了300多种罕见的遗传骨疾病，但是这些疾病的治疗通常受到限制，因为他们的病原体知之甚少。罕见疾病的研究受到原代细胞/组织的无法获得和缺乏合适的动物模型的困扰。最近在患者特异性诱导的多能茎（IPS）细胞生物学方面进步开辟了用于研究患者骨细胞的新途径。在此应用中，我计划使用患者特异性的IPS细胞研究颅骨术发育不全（CMD）的疾病机制，重点是破骨细胞（OCS），骨分辨细胞的作用。 CMD的发作始于童年时期，颅面增厚和异常形状的长骨头开始。它的终生进展会导致某些患者威胁生命的后果。迄今为止，除了重复手术外，没有其他治疗方法。先前在带有CMD引起ANK突变的敲入（KL）小鼠模型中的研究表明，ANK [Ki/Ki]小鼠的OC缺陷。在CMD患者的人外周血培养物中发现了类似的结果。 ANK [Ki/ki] OCS还显示出异常的肌动蛋白组织运动较慢。提出了两个具体的目的，以检验以下假设，即通过负面影响肌动蛋白的细胞骨架来减少单个破骨细胞活性。在AIM 1中，申请人将比较来自健康对照和CMD患者的IPS衍生的OC，以确定OC形成，基质和矿物吸收的差异，表达 OC标记基因，粘附和通过粘附测定和活细胞延时成像的迁移。在AIM 2中，申请人将通过共聚焦显微镜研究CMD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种关键调节机制，GTPase家族成员RAC，RHO和CDC42的激活以及IPS衍生的对照组和CMD患者的酪氨酸磷酸化的激活将通过主动GTPase PARLEWN分析，免疫抑制和免疫印记进行检查。或者，我们将使用FRET（基于荧光共振能量转移）分析来确定CMD患者和对照组的生命破骨细胞中RAC，RHO和CDC42的动态调节。这项研究的长期目标是将CMD建立为研究罕见遗传骨骼疾病机制的范式。