Induced Pluripotent Stem Cells in Canine Muscular Dystrophy

描述（由申请人提供）：杜氏肌营养不良症（DMD）是一种由肌营养不良蛋白基因突变引起的x连锁遗传疾病，每年在美国出生的3300名男性中就有1人患有此病，导致毁灭性的虚弱和因心肺衰竭而过早死亡。大多数DMD患者在生命的第二个十年出现明显的心肌病，约30%的病例直接导致心力衰竭死亡。然而，对DMD患者的潜在心脏病理生理知之甚少。金毛肌肉萎缩症（GRMD）是生理和临床最接近人类疾病的动物模型。与患有DMD的人一样，受GRMD影响的狗表现为进行性心力衰竭，导致早期死亡。我们的长期目标是了解肌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，我们已经证明，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性明显下降，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）的普遍萎缩可能间接导致DMD的心脏病理。因此，有必要建立一个实验系统来研究肌营养不良蛋白缺陷的心肌细胞，而不受全身性疾病的潜在混淆影响。为此，我们建议利用多能干细胞体外分化的心肌细胞。最近的研究表明，通过相对简单的重编程步骤，可以从容易获得的细胞（如皮肤成纤维细胞或角质形成细胞）中产生诱导多能干细胞（iPS），这是一项重大的科学进步，在基础研究和应用临床医学的许多领域具有深远的影响。该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，并将这些系用作特化细胞系的来源，以便在没有系统扰动的情况下评估基因型对细胞表型的影响。我们的中心假设是心肌细胞中肌营养不良蛋白的缺失直接导致收缩功能障碍。为了验证这一假设，我们将从受GRMD影响的狗和正常的窝友身上提取iPS细胞系，分离来自这些干细胞的心脏谱系祖细胞，并开始评估GRMD肌营养不良蛋白突变对心肌细胞发育和功能的影响。我们的具体目标是：从肌营养不良蛋白缺陷（GRMD）犬和正常对照中获得iPS细胞系，并与已建立的犬胚胎干细胞（cES）细胞进行比较，确认其多能干细胞的特性。1.1. 评估生长特性和多能性相关标记，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，并确定它们是否含有代表三个胚胎胚层的细胞类型。1.3. 评估体外分化到代表所有三个胚层的细胞类型。目标2。探讨肌营养不良蛋白在心肌细胞发育中的作用。2.1. 从正常和缺乏肌营养不良蛋白的犬iPS细胞系中分离心脏祖细胞。2.2. 从正常和缺乏肌营养不良蛋白的ips衍生胚状体中分离心肌细胞。2.3. 评估从正常和缺乏肌营养蛋白的iPS细胞培养中获得的心肌细胞谱系细胞中与心脏发育和成熟相关的基因表达水平。将其与正常犬和GRMD犬心脏组织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肌营养不良细胞中收缩蛋白的结构和组织。并将这些细胞与正常犬和GRMD犬的心脏组织细胞进行比较。2.5测量犬ips衍生犬心肌细胞的动作电位记录。这些目标的成功完成将验证一种独特的GRMD模型细胞系统，它将补充体内研究，并使我们能够充分表征肌营养不良蛋白突变对心肌细胞收缩功能和其他生理方面的直接影响。我们预计，基于iPS细胞的模型将在研究骨骼肌和心肌疾病进展机制，筛选潜在的治疗药物以及为治疗DMD的新再生医学方法奠定基础方面具有很大的实用价值。

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.