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首页> 外文学位 >Modeling RASopathy-associated Cardiac Disease using Patient-Specific Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
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Modeling RASopathy-associated Cardiac Disease using Patient-Specific Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

机译：使用患者特定的人类诱导的多能干细胞衍生的心肌细胞建模与RAS病相关的心脏疾病。

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摘要

The RASopathies are a family of autosomal dominant developmental disorders due to germline mutations in the RAS/MAPK signaling pathway. Among the pleomorphic defects associated with these disorders, about 80% of RASopathy patients display cardiovascular abnormalities, with hypertrophic cardiomyopathy (HCM) being one of the most common manifestations. For one RASopathy, called cardio-facio-cutaneous syndrome (CFCS), caused primarily by activating BRAF mutations, 40% of patients develop HCM. While the RAS/MAPK pathway has been implicated in cardiac hypertrophy, the role of this pathway in HCM is still unclear. In order to better understand the role of RAS/MAPK activation in HCM, we generated a human induced pluripotent stem cell model for CFCS from three patients with activating BRAF mutations. Using cell sorting, we have developed a method to examine hiPSC-derived cell-type specific phenotypes and cellular interactions underpinning HCM. We show that purified BRAF-mutant cardiomyocytes display cellular hypertrophy with prohypertrophic gene expression and intrinsic calcium handling defects. In addition, purified BRAF-mutant fibroblast-like cells exhibit a pro-fibrotic phenotype and critically modulate cardiomyocyte hypertrophy through TGFbeta paracrine signaling. Inhibition of TGFbeta or RAS/MAPK signaling is capable of rescuing the hypertrophic phenotype. Thus, we show for the first time that cell autonomous and non-autonomous defects underlie RASopathy-associated HCM, as has been demonstrated in non-syndromic HCM models. TGFbeta inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies.;Secondarily, we have developed a method for the purification of hiPSC-derived atrial-like cardiomyocytes, as there is currently no reliable method for stem cell-derived atrial cell labeling and purification. We have created a bacterial artificial chromosome (BAC) reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN). When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find utility in the study of atrial-specific pathologies and chamber-specific lineage development.
机译：RASopathies是由于RAS / MAPK信号传导途径中的种系突变而导致的常染色体显性发育障碍家族。在与这些疾病相关的多形性缺陷中，约80％的RAS病患者表现出心血管异常，其中肥厚型心肌病（HCM）是最常见的表现之一。对于一种主要由激活BRAF突变引起的称为心脏筋膜皮肤综合征（CFCS）的RAS病，40％的患者会发展为HCM。尽管RAS / MAPK途径与心脏肥大有关，但该途径在HCM中的作用仍不清楚。为了更好地了解RAS / MAPK激活在HCM中的作用，我们从三名激活BRAF突变的患者中生成了人诱导的CFCS多能干细胞模型。使用细胞分选，我们开发了一种方法来检查hiPSC衍生的细胞类型特异性表型和支撑HCM的细胞相互作用。我们显示纯化的BRAF突变型心肌细胞显示肥大性与肥大基因表达和内在钙处理缺陷。另外，纯化的BRAF突变的成纤维细胞样细胞表现出促纤维化表型并通过TGFβ旁分泌信号关键地调节心肌细胞肥大。抑制TGFbeta或RAS / MAPK信号传导能够挽救肥大的表型。因此，我们首次证明了细胞自主性和非自主性缺陷是RAS病相关HCM的基础，这已在非综合征性HCM模型中得到证实。由于RA病因或其他病因，抑制TGFβ可能是HCM患者的一种有用的治疗选择。其次，由于目前尚无可靠的干细胞检测方法，我们已经开发了一种纯化hiPSC衍生的心房样心肌细胞的方法。衍生的心房细胞标记和纯化。我们创建了一个细菌人工染色体（BAC）报告基因构建体，其中的荧光由心房特异性基因肌钙蛋白（SLN）的表达驱动。使用流式细胞仪纯化时，具有高荧光的细胞特异性表达心房基因，并显示出与心房心肌细胞一致的功能性钙处理和电生理特性。我们的数据表明SLN可以用作成功监测和分离hiPSC衍生的心房样心肌细胞的标志物。这些纯化的细胞可用于研究特定于心房的病理和特定于房室的血统。

著录项

  • 作者

    Josowitz, Rebecca.;

  • 作者单位

    Icahn School of Medicine at Mount Sinai.;

  • 授予单位 Icahn School of Medicine at Mount Sinai.;
  • 学科 Biology Genetics.;Biology Cell.;Health Sciences Human Development.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 151 p.
  • 总页数 151
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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