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:8028679
- 负责人:
- 金额:$ 12.1万
- 依托单位:
- 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:
- 关键词:ActinsAdhesionsAffectAlbers-Schonberg diseaseAnimal ModelAreaBiological AssayBiologyBloodBone DiseasesBone MarrowCellsCharacteristicsChildhoodConfocal MicroscopyCytoskeletonDataDefectDental PulpDental SchoolsDentistsDiseaseDysplasiaEnvironmentFamily memberFibroblastsFlareGenderGene ExpressionGenesGeneticGoalsGuanosine Triphosphate PhosphohydrolasesHumanHuman GeneticsHyperostosisImageImage AnalysisImmigrationImmunoblottingIndividualInvestigationKnock-in MouseLifeMedicineMentorsMethodsModificationMolecularMovementMutationOperative Surgical ProceduresOrphan DiseaseOsteoclastsOsteoporosisPathogenesisPatientsPhasePlagueRare DiseasesRegulationResearchReverse Transcriptase Polymerase Chain ReactionScientistShapesSkinSpecimenStaining methodStainsStem cellsSystemTechniquesTestingTimeTissuesTrainingTyrosine Phosphorylationabstractingbasebonebone cellcareercell motilitycellular imagingcraniofacialdeciduous tootheffective therapyhuman stem cellsimprovedinduced pluripotent stem cellinterestlong bonemigrationmouse modelnew therapeutic targetnovelperipheral bloodpluripotencyresearch studyrhosexskeletal disorderstemstem cell biologytool
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):已鉴定出 300 多种罕见的遗传性骨病,但这些疾病的治疗通常受到限制,因为对其发病机制知之甚少。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,从患者身上重复采集标本通常是不切实际的,而且可能是不道德的,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(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 细胞来研究颅骨干骺端发育不良 (CMD),重点关注破骨细胞 (OC),即骨吸收细胞。 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 (KI) 小鼠模型的研究揭示了 AnkKI/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。 AnkKI/KI OCs 还表现出运动较慢且肌动蛋白组织异常。为了检验 CMD 小鼠模型产生的假设,即引起 CMD 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,提出了四个具体目标。在目标1和2(K99阶段)中,申请人将优化方法1)从对照个体和CMD患者的成纤维细胞或SHED(来自人类脱落乳牙的干细胞)生成iPS细胞和2)将iPS细胞分化为OC 。在目标 3(R00 阶段)中,申请人将比较来自健康对照和 CMD 患者的 iPS 衍生 OC,以通过 TRAP 染色、吸收坑测定、真实-识别 OC 形成、功能、OC 标记基因表达、粘附和迁移来识别 OC 的差异。分别是时间PCR、粘附测定和活细胞延时成像。在目标 4(R00 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,以及对照和 CMD OC 中 ANK 和肌动蛋白的共定位。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 下拉测定、免疫染色和免疫印迹来检查。本研究的最终目标是以 CMD 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 牙科医学院的主要兴趣领域。研究环境非常适合执行该项目并发展申请人在牙医/科学家领域的职业生涯。
公共健康相关性:该应用程序使用人类诱导多能干 (iPS) 细胞来研究颅骨干骺端发育不良 (CMD) 中的破骨细胞缺陷,这是一种罕见的遗传性骨病。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,这可能有益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(如骨质疏松症或骨石症)的研究。
项目成果
期刊论文数量(0)
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I-Ping Chen其他文献
I-Ping Chen的其他文献
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{{ truncateString('I-Ping Chen', 18)}}的其他基金
Pathogenic Mechanisms of Craniometaphyseal Dysplasia
颅骨干骺端发育不良的发病机制
- 批准号:
10444184 - 财政年份:2022
- 资助金额:
$ 12.1万 - 项目类别:
Pathogenic Mechanisms of Craniometaphyseal Dysplasia
颅骨干骺端发育不良的发病机制
- 批准号:
10630298 - 财政年份:2022
- 资助金额:
$ 12.1万 - 项目类别:
Quantitative Assessment of Dental Pain using a smartphone-attachable electrodermal activity sensor
使用智能手机连接的皮肤电活动传感器对牙痛进行定量评估
- 批准号:
10171570 - 财政年份:2020
- 资助金额:
$ 12.1万 - 项目类别:
Pathogenic Mechanisms of Craniometaphyseal Dysplasia
颅骨干骺端发育不良的发病机制
- 批准号:
9294024 - 财政年份:2016
- 资助金额:
$ 12.1万 - 项目类别:
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
- 批准号:
8532878 - 财政年份:2012
- 资助金额:
$ 12.1万 - 项目类别:
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
- 批准号:
8508552 - 财政年份:2012
- 资助金额:
$ 12.1万 - 项目类别:
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
- 批准号:
8721393 - 财政年份:2012
- 资助金额:
$ 12.1万 - 项目类别:
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
- 批准号:
8143488 - 财政年份:2010
- 资助金额:
$ 12.1万 - 项目类别:
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