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

描述（由申请人提供）：干细胞被认为在未来几十年内有很大的希望用于治疗先天性疾病。众所周知，胚胎干细胞（ES）具有分化成广谱细胞类型（多能性）的能力，它还分泌纠正因子，防止致命的先天性心脏缺陷的发生。使用小鼠模型的“薄心肌综合征”（Id敲除小鼠），我们已经表明，分泌因子从ES细胞正常化基因表达谱的邻居细胞。ES细胞不仅可以注射到Id KO胚泡中，而且可以注射到将携带Id KO胚胎的雌性小鼠中，从而挽救先天性心脏缺陷。ES细胞在注射到mdx（杜氏肌营养不良症，DMD的小鼠模型）胚泡中时也可以挽救肌营养不良症。在这种情况下，救援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。胚胎干细胞治疗人类疾病的潜在用途受到了围绕破坏人类卵母细胞或受精胚胎的伦理问题的影响。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，小鼠体细胞去分化为胚胎干细胞样状态的转录因子的掺入。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（镰状细胞贫血）。在本申请中，我们希望表征鼠iPS细胞在挽救心脏和骨骼肌疾病中的作用，例如Id敲除小鼠和mdx小鼠。为此，我们将小鼠iPS细胞注射到小鼠胚泡中（Id KO和mdx），腹膜内注射到将携带突变胚胎的雌性小鼠中（Id KO），以及腹膜内注射到易患扩张型心肌病的小鼠中（Id条件性KO）。我们假设iPS细胞将拯救Id KO胚胎的心脏表型以及mdx小鼠的肌营养不良症。将评价Id KO胚胎的存活率。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）水平上评价校正。将确定薄心肌综合征（Id）挽救中潜在挽救分子的分泌和肌营养不良症（mdx）挽救中肌营养不良蛋白的扩散。这些实验将有助于阐明iPS细胞可能用于影响肌肉校正的机制，并将扩大iPS细胞的治疗适用性。公共卫生相关性：胚胎干细胞具有分化为所有细胞类型和释放愈合因子的独特能力。这一特点使它们成为再生医学竞技场的中心。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，必须找到替代品，使细胞同样有效。最近，诱导多能干细胞（iPS）成为最好的替代品。iPS细胞在用于表征ES细胞（包括形成小鼠嵌合体的能力）的最严格的测定中表型复制ES细胞。由于iPS细胞的产生不涉及使用胚胎，因此消除了伦理问题。为了评估iPS细胞的治疗潜力，测试iPS细胞在纠正各种遗传疾病中的作用将是非常重要的。在这些应用中，我们提出在两种肌肉疾病（心脏和骨骼肌）中挑战鼠iPS细胞，我们表明这两种肌肉疾病可以通过ES细胞治疗来纠正。通过ES细胞纠正这两种疾病的机制是不同的-在一种情况下分泌因子，而在另一种情况下传播结构蛋白。我们计划将iPS细胞注射到易患这些疾病的早期胚胎中。我们还计划在受孕前将iPS细胞注射到雌性小鼠体内。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，我们希望iPS细胞将防止病理发生。这些实验将有助于阐明iPS细胞可用于在心肌和骨骼肌中实现校正的分子机制，并将拓宽iPS细胞的治疗适用性。

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.