Role of murine induced pluripotent stem cells on the correction of cardiac and sk

小鼠诱导多能干细胞对心脏和骨骼肌校正的作用

• 批准号: 7888348
• 负责人: DIEGO FRAIDENRAICH
• 金额: $ 23.4万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7888348
• 关键词: Adenovirus Vector; Adult; Age-Months; Alleles; Animals; Area; Autologous; Biological Assay; Cardiac; Cardiomyopathies; Cells; Chimera organism; Chimerism; Clinic; Clinical; Communities; Complex; Conceptions; Congenital Heart Defects; DNA; Defect; Detection; Dilated Cardiomyopathy; Disease; Duchenne muscular dystrophy; Dystrophin; Echocardiography; Embryo; Ethical Issues; Ethics; Female; Fiber; Gene Expression; Generations; Genes; Genotype; Germ Layers; Germ Lines; Glycoproteins; Goals; Healed; Heart; Hematopoietic; Hereditary Disease; Histologic; Histology; Human; Hypertrophy; IGF1 gene; Immunofluorescence Immunologic; Immunohistochemistry; Implant; Inflammatory; Injection of therapeutic agent; Intraperitoneal Injections; Knockout Mice; LacZ Genes; Methylation; Modality; Modeling; Molecular; Molecular Profiling; Mus; Muscle; Muscle Fibers; Muscular Dystrophies; Mutate; Mutation; Myocardial; Myocardium; Myopathy; Myosin Heavy Chains; Names; Newborn Infant; Oocytes; Pathology; Pathway interactions; Phenocopy; Phenotype; Play; Pregnancy; Production; Proteins; Regenerative Medicine; Role; Sickle Cell Anemia; Signal Transduction; Silver; Skeletal Muscle; Somatic Cell; Southern Blotting; Staining method; Stains; Stem cells; Structural Protein; Structure; Survival Analysis; Syndrome; Tail; Testing; Therapeutic; Tissues; Transplantation; Treadmill Tests; Ursidae Family; Utrophin; Ventricular Septal Defects; Western Blotting; blastocyst; c-myc Genes; cell type; dystrobrevin; embryonic stem cell; gene correction; healing; hemodynamics; human disease; induced pluripotent stem cell; mdx mouse; mouse model; mutant; pluripotency; postnatal; prevent; progenitor; public health relevance; pup; research study; stem; syntrophin; transcription factor

DESCRIPTION (provided by applicant): Stem cells are regarded with great promise in the next decades for treatment of congenital disease. Well known for their capacity to differentiate into a broad spectrum of cell types (pluripotency), embryonic stem (ES) cells also secrete corrective factors that prevent lethal congenital heart defects from occurring. Using a mouse model of the "thin myocardial syndrome" (Id knockout mice), we have shown that secreted factors from ES cells normalize gene expression profiles in neighbor cells. The ES cells can rescue congenital heart defects not only when injected into Id KO blastocysts but also into mouse females that will bear Id KO embryos. The ES cells can also rescue muscular dystrophy when injected into mdx (a mouse model of Duchenne muscular dystrophy, DMD) blastocysts. Rather than factor secretion, the main mechanism of the rescue in this case is spreading of ES-derived dystrophin (the protein absent in DMD) throughout most of the musculature to stabilize the muscle. The potential use of ES cells to treat human disease is clouded by ethical concerns surrounding the destruction of human oocytes or fertilized embryos. The scientific community is looking for alternatives that would not use embryos as starting material. Recent experiments demonstrated that murine somatic cells de-differentiate to an embryonic stem cell-like status by the incorporation of transcription factors. These cells were named induced pluripotent stem cells (iPS cells). The iPS cells are similar to the ES cells. Remarkably, the generation of iPS cells does not require destruction of embryos, therefore there are no ethical concerns. In addition, a recent proof-of-principle experiment showed that iPS cells can correct disease (sickle cell anemia) in mice. In this application we would like to characterize the murine iPS cells in their role to rescue cardiac and skeletal muscle disease, exemplified by the Id knockout mice and the mdx mice. To this end, we will inject murine iPS cells into murine blastocysts (Id KO and mdx), intraperitoneally into female mice that will harbor mutant embryos (Id KO), and also intraperitoneally into mice predisposed to develop dilated cardiomyopathy (Id conditional KO). We hypothesize that iPS cells will rescue the cardiac phenotype of the Id KO embryos as well as muscular dystrophy in mdx mice. Survival of the Id KO embryos will be evaluated. Corrections will be evaluated at the histological (immunohistochemistry, H&E, immunofluorescence) and functional (echocardiography, treadmill) level. Secretion of potential rescue molecules in the thin myocardial syndrome (Id) rescue and spreading of dystrophin in the muscular dystrophy (mdx) rescue will be determined. These experiments will help elucidate the mechanisms that the iPS cells may utilize to effect corrections in muscle and will broaden the therapeutic applicability of the iPS cells. PUBLIC HEALTH RELEVANCE: ES cells have the unique capacity to differentiate into all cell types and to emit healing factors. This feature places them at the center of the regenerative medicine arena. Technical and ethical issues compromise the enthusiasm for the use of ES cells in a clinical setting. Therefore it becomes imperative to find alternatives that will render cells equally potent. Recently the induced pluripotent stem (iPS) cells emerged as the greatest alternative. The iPS cells phenocopy the ES cells in the most rigorous assays that are used to characterize the ES cells, including the capacity to form mouse chimeras. As the generation of iPS cells does not involve the use of embryos, the ethical concerns are eliminated. To assess the therapeutic potential of the iPS cells, it will be extremely important to test the role that the iPS cells play in correcting a variety of genetic diseases. In these application we propose to challenge the murine iPS cells in two muscular diseases (heart and skeletal muscle) that we showed can be corrected by ES cell treatment. The mechanism of the correction of both diseases by the ES cells is different - in one case secretion of factors and in another spreading of a structural protein. We plan to inject iPS cells into early embryos that are predisposed to develop these diseases. We also plan to inject the iPS cells into female mice before conception. Finally, we plan to inject the iPS cells into mice predisposed to develop dilated cardiomyopathy. In all cases we hope the iPS cells will prevent pathology from occurring. These experiments will help elucidate the molecular mechanisms that the iPS cells may utilize to effect corrections in cardiac and skeletal muscle and will broaden the therapeutic applicability of the iPS cells.

描述（由申请人提供）：干细胞被认为在未来几十年内有望用于治疗先天性疾病。胚胎干细胞 (ES) 以其分化为多种细胞类型（多能性）的能力而闻名，它们还分泌纠正因子，防止致命的先天性心脏缺陷的发生。使用“薄心肌综合征”小鼠模型（Id 基因敲除小鼠），我们发现 ES 细胞分泌的因子使邻近细胞的基因表达谱正常化。 ES细胞不仅可以注射到Id KO囊胚中，还可以注射到将产生Id KO胚胎的雌性小鼠中，从而挽救先天性心脏缺陷。当将 ES 细胞注射到 mdx（杜氏肌营养不良症 (DMD) 小鼠模型）囊胚中时，ES 细胞还可以挽救肌营养不良症。在这种情况下，拯救的主要机制不是因子分泌，而是 ES 衍生的肌营养不良蛋白（DMD 中不存在的蛋白质）在大部分肌肉组织中扩散，以稳定肌肉。 ES细胞治疗人类疾病的潜在用途因破坏人类卵母细胞或受精胚胎的伦理问题而蒙上阴影。科学界正在寻找不使用胚胎作为起始材料的替代品。最近的实验表明，小鼠体细胞通过掺入转录因子而去分化为胚胎干细胞样状态。这些细胞被命名为诱导多能干细胞（iPS 细胞）。 iPS 细胞与 ES 细胞相似。值得注意的是，iPS 细胞的产生不需要破坏胚胎，因此不存在伦理问题。此外，最近的一项原理验证实验表明，iPS 细胞可以纠正小鼠的疾病（镰状细胞性贫血）。在此应用中，我们希望描述小鼠 iPS 细胞在拯救心脏和骨骼肌疾病方面的作用，以 Id 敲除小鼠和 mdx 小鼠为例。为此，我们将小鼠 iPS 细胞注射到小鼠囊胚（Id KO 和 mdx）中，腹膜内注射到将携带突变胚胎的雌性小鼠（Id KO）中，并且还腹膜内注射到易患扩张型心肌病的小鼠（Id 条件 KO）中。我们假设 iPS 细胞将挽救 Id KO 胚胎的心脏表型以及 mdx 小鼠的肌营养不良症。将评估 Id KO 胚胎的存活率。校正将在组织学（免疫组织化学、H&E、免疫荧光）和功能（超声心动图、跑步机）水平上进行评估。将确定在薄心肌综合征（Id）救援中潜在救援分子的分泌以及在肌营养不良（mdx）救援中抗肌营养不良蛋白的传播。这些实验将有助于阐明 iPS 细胞可用于影响肌肉校正的机制，并将扩大 iPS 细胞的治疗适用性。公共健康相关性：ES 细胞具有分化成所有细胞类型并释放治疗因子的独特能力。这一特征使它们处于再生医学领域的中心。技术和伦理问题损害了在临床环境中使用 ES 细胞的热情。因此，必须找到使细胞具有同等效力的替代品。最近，诱导多能干（iPS）细胞成为最好的选择。 iPS 细胞通过最严格的检测对 ES 细胞进行表型复制，这些检测用于表征 ES 细胞，包括形成小鼠嵌合体的能力。由于 iPS 细胞的产生不涉及胚胎的使用，因此消除了伦理问题。为了评估 iPS 细胞的治疗潜力，测试 iPS 细胞在纠正多种遗传疾病中的作用极其重要。在这些应用中，我们建议在两种肌肉疾病（心脏和骨骼肌）中挑战小鼠 iPS 细胞，我们证明这两种疾病可以通过 ES 细胞治疗来纠正。 ES细胞纠正这两种疾病的机制是不同的——一种是分泌因子，另一种是传播结构蛋白。我们计划将 iPS 细胞注入易患这些疾病的早期胚胎中。我们还计划在雌性小鼠受孕前将 iPS 细胞注射到体内。最后，我们计划将 iPS 细胞注射到易患扩张型心肌病的小鼠体内。在所有情况下，我们都希望 iPS 细胞能够防止病理发生。这些实验将有助于阐明 iPS 细胞可用于影响心肌和骨骼肌校正的分子机制，并将扩大 iPS 细胞的治疗适用性。

项目成果

Differential requirement for utrophin in the induced pluripotent stem cell correction of muscle versus fat in muscular dystrophy mice.

肌营养不良症小鼠诱导多能干细胞校正肌肉与脂肪时对肌营养不良蛋白的差异需求。

• 发表时间: 2011
• 影响因子: 3.7
• 作者: Beck, Amanda J;Vitale, Joseph M;Zhao, Qingshi;Schneider, Joel S;Chang, Corey;Altaf, Aneela;Michaels, Jennifer;Bhaumik, Mantu;Grange, Robert;Fraidenraich, Diego
• 通讯作者: Fraidenraich, Diego