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）：在未来几十年治疗先天性疾病中，干细胞被认为具有很大的希望。胚胎茎（ES）细胞以分化为广泛细胞类型（多能性）的能力而闻名，还分泌矫正因子，以防止致命的先天性心脏缺陷发生。使用“薄心肌综合征”（ID基因敲除小鼠）的小鼠模型，我们表明来自ES细胞的分泌因子使邻居细胞中的基因表达谱正常化。 ES细胞不仅可以在注射ID KO胚泡，还可以挽救先天性心脏缺陷，还可以挽救将带有ID KO胚胎的小鼠雌性。当注射到MDX（Duchenne肌肉营养不良的小鼠模型，DMD）中时，ES细胞还可以挽救肌肉营养不良。在这种情况下，救援的主要机制不是因素分泌，而是在大多数肌肉组织中传播ES衍生的肌营养不良蛋白（DMD中缺乏蛋白质）以稳定肌肉。 ES细胞来治疗人类疾病的潜在用途是由于围绕人类卵母细胞或受精胚胎的破坏而引起的。科学界正在寻找不会使用胚胎作为起始材料的替代方法。最近的实验表明，通过掺入转录因子，鼠体细胞细胞脱离了胚胎干细胞状态。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，因此没有道德问题。此外，最近的原则实验表明，IPS细胞可以纠正小鼠中的疾病（镰状细胞贫血）。在此应用中，我们想表征鼠IPS细胞在挽救心脏和骨骼肌疾病中的作用中的作用，以ID敲除小鼠和MDX小鼠为例。为此，我们将将鼠IPS细胞注入鼠胚泡（ID KO和MDX）中，将腹膜内腹膜内注入雌性小鼠中，该小鼠将含有突变胚胎（ID KO），并腹膜内腹膜内腹膜内，将其倾向于产生扩张的心肌病（ID条件KO）。我们假设IPS细胞将营救ID KO胚胎的心脏表型以及MDX小鼠的肌肉营养不良。 ID KO胚胎的存活率将进行评估。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。将确定肌营养不良症（MDX）救援中肌营养不良蛋白的稀薄心肌综合征（ID）中的潜在救援分子的分泌。这些实验将有助于阐明IPS细胞可以用来实现肌肉校正的机制，并将扩大IPS细胞的治疗适用性。公共卫生相关性：ES细胞具有区分所有细胞类型并发出治愈因素的独特能力。此功能将它们置于再生医学领域的中心。技术和道德问题损害了在临床环境中使用ES细胞的热情。因此，必须找到可以使细胞同样有效的替代方法。最近，诱导的多能干（IPS）细胞成为最大的选择。 IPS细胞在最严格的测定中表征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，因此消除了道德问题。为了评估IPS细胞的治疗潜力，测试IPS细胞在纠正各种遗传疾病中起起作用的作用将非常重要。在这些应用中，我们建议在我们表现出的两种肌肉疾病（心脏和骨骼肌）中挑战鼠IPS细胞，可以通过ES细胞处理来纠正。 ES细胞对两种疾病进行纠正的机制都是不同的 - 在一种情况下，因素分泌以及结构蛋白的另一种扩散。我们计划将IPS细胞注入早期的胚胎，这些胚胎易于发展这些疾病。我们还计划在受孕之前将IPS细胞注入雌性小鼠。最后，我们计划将IPS细胞注射到易感性心肌病的小鼠中。在所有情况下，我们希望IPS细胞都可以防止病理发生。这些实验将有助于阐明IPS细胞可以用来实现心脏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.

• DOI: 10.1371/journal.pone.0020065
• 发表时间: 2011
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Beck AJ;Vitale JM;Zhao Q;Schneider JS;Chang C;Altaf A;Michaels J;Bhaumik M;Grange R;Fraidenraich D
• 通讯作者: Fraidenraich D