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治疗先天性疾病有很大的希望。众所周知，胚胎干细胞具有分化为多种细胞类型（多能性）的能力，它们还能分泌校正因子，防止致命的先天性心脏缺陷的发生。利用小鼠模型的“瘦心肌综合征”（Id敲除小鼠），我们已经表明，从胚胎干细胞分泌因子正常化基因表达谱在邻近细胞。胚胎干细胞不仅可以被注射到Id KO囊胚中，还可以被注射到将产生Id KO胚胎的雌性小鼠中，从而挽救先天性心脏缺陷。将胚胎干细胞注入mdx（小鼠杜氏肌营养不良症模型，DMD）囊胚中，也能挽救肌营养不良。在这种情况下，救援的主要机制不是因子分泌，而是es来源的肌营养不良蛋白（DMD中缺乏的蛋白质）在大部分肌肉组织中扩散，以稳定肌肉。胚胎干细胞用于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，小鼠体细胞通过转录因子的掺入向胚胎干细胞样状态去分化。这些细胞被命名为诱导多能干细胞（iPS cells）。iPS细胞与胚胎干细胞相似。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）。在这个应用中，我们想要描述小鼠iPS细胞在拯救心脏和骨骼肌疾病中的作用，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，腹腔注射到携带突变胚胎的雌性小鼠（Id KO）中，也腹腔注射到易患扩张性心肌病的小鼠（Id条件型KO）中。我们假设iPS细胞将拯救Id KO胚胎的心脏表型以及mdx小鼠的肌肉萎缩症。将评估Id KO胚胎的存活情况。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）水平上评估纠正。确定瘦心肌综合征（Id）抢救中潜在抢救分子的分泌和肌营养不良（mdx）抢救中肌营养不良蛋白的扩散。这些实验将有助于阐明iPS细胞可能用于肌肉矫正的机制，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。这一特点使他们处于再生医学舞台的中心。技术和伦理问题削弱了在临床环境中使用胚胎干细胞的热情。因此，必须找到能使细胞具有同等效力的替代品。最近，诱导多能干细胞（iPS）成为最大的替代方法。iPS细胞在最严格的测定中表现出胚胎干细胞的表型，这些测定用于表征胚胎干细胞，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细胞在纠正多种遗传疾病中的作用将是极其重要的。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，我们发现这两种疾病可以通过胚胎干细胞治疗得到纠正。胚胎干细胞纠正这两种疾病的机制是不同的——在一种情况下是因子的分泌，在另一种情况下是结构蛋白的扩散。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细胞能够防止病理发生。这些实验将有助于阐明诱导多能干细胞可能用于心脏和骨骼肌校正的分子机制，并将扩大诱导多能干细胞的治疗适用性。

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.