Induced Pluripotent Stem Cells in Canine Muscular Dystrophy

诱导多能干细胞治疗犬肌营养不良症

• 批准号: 8067098
• 负责人: Martin K Childers
• 金额: $ 23.46万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8067098
• 关键词: Action Potentials; Adrenergic Agents; Adult; Affect; Animal Model; Area; Atrophic; Award; Basic Science; Biopsy; Canis familiaris; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Line; Cell Lineage; Cell model; Cells; Cessation of life; Clinical Medicine; Complement; Congenital Heart Defects; Contractile Proteins; Depressed mood; Development; Disease; Disease Progression; Duchenne muscular dystrophy; Dystrophin; Embryo; Experimental Models; Failure; Fibroblasts; Functional disorder; Gene Mutation; Genes; Genotype; Germ Layers; Goals; Growth; Heart; Heart failure; Hereditary Disease; Human; Immune; In Vitro; K-18 conjugate; Life; Link; Measures; Methods; Modeling; Molecular; Mus; Muscular Dystrophies; Mutation; Myocardium; Normal tissue morphology; Pathology; Patients; Phenotype; Physiology; Pluripotent Stem Cells; Positioning Attribute; Property; Regenerative Medicine; Respiration; Role; Scientific Advances and Accomplishments; Seminal; Skeletal Muscle; Skin; Source; Stem Cell Research; Stem cells; Structure; Surface Antigens; System; Systemic disease; Technology; Telomerase; Teratoma; Testing; Therapeutic Agents; Tissues; adrenergic; base; career; cell type; disease phenotype; embryonic stem cell; human disease; in vivo; induced pluripotent stem cell; keratinocyte; male; mutant; novel; pluripotency; progenitor; public health relevance; response; stem; transcription factor; tumor

DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD), an X-linked genetic disorder resulting from mutations in the dystrophin gene, afflicts 1 in 3300 males born each year in the US, causing devastating weakness and early death from cardiorespiratory failure. Most DMD patients develop pronounced cardiomyopathy by the second decade of life, with heart failure directly leading to death in about 30% of cases. However, little is known about the underlying cardiac pathophysiology in DMD patients. Golden retriever muscular dystrophy (GRMD) is the animal model that physiologically and clinically most closely resembles the human disease. Like humans with DMD, affected GRMD dogs display progressive heart failure leading to early death. Our long-term goal is to understand the cellular and molecular mechanisms by which dystrophin deficiency leads to cardiac abnormalities. In preliminary studies we have demonstrated markedly depressed contractility and impaired beta-adrenergic responsiveness in isolated cardiomyocytes from heart tissue of GRMD dogs compared with unaffected controls. However, the effects of general atrophy of skeletal muscles, including those supporting respiration, may indirectly contribute to the cardiac pathology in DMD. Therefore, it is imperative to develop an experimental system to study dystrophin deficient cardiomyocytes without the potential confounding effects of systemic disease. For this purpose we propose to utilize cardiomyocytes differentiated in vitro from pluripotent stem cells. The recent demonstration that it is possible to generate induced pluripotent stem (iPS) cells from easily obtained cells such as skin fibroblasts or keratinocytes, by relatively simple reprogramming steps, constitutes a major scientific advance with profound ramifications in many areas of basic research and applied clinical medicine. A powerful application of this technology is to obtain stem cell lines carrying medically significant gene mutations and to use these lines as a source of specialized cell lineages in order to assess effects of genotype on cellular phenotype in the absence of systemic perturbations. Our central hypothesis posits that loss of dystrophin in cardiomyocytes results directly in contractile dysfunction. To test this premise we will produce iPS cell lines from affected GRMD dogs and normal littermates, isolate cardiac lineage progenitors derived from these stem cells, and initiate assessment of the influence of the GRMD dystrophin mutation on cardiomyocyte development and function. Our Specific Aims are: Aim 1. To generate iPS cell lines from dystrophin-deficient (GRMD) dogs and normal controls, and confirm their character as pluripotent stem cells, in comparison with established canine embryonic stem (cES) cells. 1.1. Assess growth properties and pluripotency-associated markers, including cell surface antigens, transcription factors and telomerase. 1.2. Assess formation of teratoma tumors in immune deficient mice, and determine whether they contain cell types representative of the three embryonic germ layers. 1.3. Assess differentiation in vitro to cell types representative of all three germ layers. Aim 2. To examine the role of dystrophin in cardiomyocyte development. 2.1. Isolate cardiac progenitors from normal and dystrophin deficient canine iPS cell lines. 2.2. Isolate cardiomyocytes from normal and dystrophin deficient iPS-derived embryoid bodies. 2.3. Assess the expression levels of genes associated with cardiac development and maturation in cardiomyocyte lineage cells obtained in culture from normal and dystrophin-deficient iPS cells. Compare these with expression in cardiomyocytes from heart tissue of normal and GRMD dogs. 2.4. Compare the structure and organization of contractile proteins in developing normal and dystrophin- deficient cardiomyocytes generated in vitro from iPS cells. Also compare these with cells from cardiac tissue of normal and GRMD dogs. 2.5 Measure action potential recordings of canine iPS-derived canine cardiomyoctyes. The successful completion of these Aims will validate a unique GRMD model cell system that will complement in vivo studies and position us to fully characterize the direct effects of a dystrophin mutation on cardiomyocyte contractile function and other aspects of physiology. We anticipate that the iPS cell-based model will have great utility to study mechanisms of disease progression in skeletal and cardiac muscle, to screen for potential therapeutic agents, and to lay the groundwork for novel regenerative medicine approaches to treat DMD. PUBLIC HEALTH RELEVANCE: We hypothesize that the loss of dystrophin in cardiomyocytes causes a disease phenotype at the cellular level. We will test this premise in cardiomyocytes isolated from induced pluripotent stem cell lines derived from dogs harboring a dystrophin mutation.

描述(申请人提供)：Duchenne肌营养不良症(DMD)是一种X连锁遗传病，由dystrophin基因突变引起，每年在美国出生的3300名男性中就有1人患有此病，导致严重的虚弱和心肺衰竭早期死亡。大多数DMD患者在生命的第二个十年会出现明显的心肌病，大约30%的病例会直接导致心力衰竭。然而，对DMD患者潜在的心脏病理生理学知之甚少。金毛猎犬肌营养不良症(GRMD)是一种在生理和临床上与人类疾病最为相似的动物模型。与患有DMD的人类一样，受影响的GRMD狗表现出进行性心力衰竭，导致过早死亡。我们的长期目标是了解营养不良蛋白缺乏导致心脏异常的细胞和分子机制。在初步研究中，我们发现，与未受影响的对照组相比，GRMD犬心脏组织中分离的心肌细胞的收缩能力明显降低，β-肾上腺素能反应性受损。然而，骨骼肌包括支持呼吸的骨骼肌萎缩的影响可能间接地促进了DMD的心脏病理。因此，有必要建立一个实验系统来研究dystrophin缺陷型心肌细胞，而不受全身性疾病的潜在混杂影响。为此，我们建议利用从多能干细胞在体外分化的心肌细胞。最近证明，通过相对简单的重新编程步骤，可以从皮肤成纤维细胞或角质形成细胞等容易获得的细胞中产生诱导多能干细胞(IPS)，这是一项重大的科学进步，对基础研究和应用临床医学的许多领域产生了深远的影响。这项技术的一个强大应用是获得携带医学上重要基因突变的干细胞系，并将这些干细胞系用作特殊细胞谱系的来源，以便在没有系统性干扰的情况下评估基因对细胞表型的影响。我们的中心假设是，心肌细胞中dystrophin的缺失直接导致收缩功能障碍。为了测试这一前提，我们将从受GRMD影响的狗和正常胎鼠中培养iPS细胞系，分离来自这些干细胞的心脏祖细胞，并开始评估GRMD dystrophin突变对心肌细胞发育和功能的影响。目的：1.从营养不良蛋白缺陷型(GRMD)犬和正常对照犬中建立iPS细胞系，并与已建立的犬胚胎干细胞(CES)进行比较，证实其为多能干细胞。1.1.评估生长特性和多能性相关标记物，包括细胞表面抗原、转录因子和端粒酶。1.2.评估免疫缺陷小鼠畸胎瘤肿瘤的形成，并确定它们是否包含代表三个胚胎生殖层的细胞类型。1.3.评估在体外对代表所有三种生殖层的细胞类型的分化。目的2.研究dystrophin在心肌细胞发育中的作用。2.1.从正常和dystrophin缺陷型犬iPS细胞系中分离心脏祖细胞。2.2.从正常和dystrophin缺陷的iPS来源的类胚体中分离心肌细胞。2.3.评估从正常和dystrophin缺陷的iPS细胞中获得的心肌细胞系细胞中与心脏发育和成熟相关的基因的表达水平。将这些结果与正常和GRMD犬心脏组织中心肌细胞的表达进行比较。2.4.比较iPS细胞体外生成的正常心肌细胞和dystrophin缺陷型心肌细胞中收缩蛋白的结构和组织。并将这些细胞与正常和GRMD犬的心脏组织细胞进行比较。2.5测量犬iPS来源的犬心肌的动作电位记录。这些目标的成功完成将验证一个独特的GRMD模型细胞系统，该系统将补充体内研究，并使我们能够充分表征dystrophin突变对心肌细胞收缩功能和其他生理学方面的直接影响。我们预计，基于iPS细胞的模型将对研究骨骼肌和心肌疾病进展的机制，筛选潜在的治疗药物，以及为治疗DMD的新的再生医学方法奠定基础。 公共卫生相关性：我们假设心肌细胞中dystrophin的缺失会导致细胞水平的疾病表型。我们将在从诱导多能干细胞系中分离的心肌细胞中测试这一前提，这些干细胞系来自携带dystrophin突变的狗。