Identical Twins Discordant for Juvenile Dermatomyositis: iPSC-Myogenic Cells

描述（由申请人提供）：青少年皮肌炎是最常见的儿童炎症性肌病，是一种全身性儿童自身免疫性疾病。目前还没有JDM的实验模型，这极大地限制了我们对疾病过程的病理生理学的理解，与其他自身免疫性疾病一样，JDM与过早的心血管损害和慢性炎症有关。可遗传的遗传因素，无论是独特的还是与其他自身免疫性疾病共有的，都与环境影响（如UVB暴露、出生季节和先前感染）相一致，使儿童易患JDM。肌肉特异性分子途径的表征受到可用于研究的诊断活检所涉及的肌肉数量有限的严重限制。我们已经记录了未经治疗的JDM儿童肌肉中mRNA水平的失调和miRNA控制的异常，以及肌纤维本身活跃的细胞因子的产生，这表明它们参与免疫反应，而不仅仅是免疫反应的目标。最近的一项发现表明，存在于少量（5ml）外周血中的单个核细胞可以通过不整合的仙台病毒感染它们来“重新编程”，从而产生“诱导多能干细胞”（iPSC）。多能干细胞与胚胎干细胞有许多共同的特征；当移植到小鼠体内时，它们可以诱导畸胎瘤的形成，并产生广泛的分化细胞类型，其中包括肌源性祖细胞，这些细胞随后发育成肌肉。在这项研究中，我们将从3对JDM不一致的同卵双胞胎及其匹配的健康对照中获得肌源性前体细胞的单细胞群，以表征肌肉对疾病病理生理的遗传贡献。我们假设，来自iPSCs的分化肌肉细胞将重现与表型相关的分子差异，我们之前在未经治疗的JDM儿童与健康对照的肌肉活检中发现了与表型相关的转录组（miRNAs和mrna）。在Specific Aim #1中，我们将从三组同卵双胞胎中获得并比较iPSCs：一组被诊断为JDM，一组未受影响，以及年龄、性别、种族匹配的对照组，以便：a)通过在小鼠宿主中形成畸胎瘤来验证其多能性，b)诱导胚胎样体细胞在体外形成骨骼肌。未受影响的双胞胎将作为特定的遗传和环境控制。特异性目标# 2将使用RNAseq和miRNA分析表征转录组差异：1)来自JDM，未受影响的双胞胎和健康匹配对照的IPSc，以及2)其IPSc衍生的肌源性细胞，以确定来自每个供体以及供体之间IPSc和肌肉的差异。生物信息学分析将使数据正常化，并在每个供体的iPSCs及其衍生的肌肉细胞中识别差异表达的基因及其途径。该项目的成功将有助于加深对炎症性肌病的认识，并为JDM和其他自身免疫性疾病的病理生理学研究开辟新的途径。

DESCRIPTION (provided by applicant): Juvenile Dermatomyositis, the most common of the pediatric inflammatory myopathies, is a systemic pediatric autoimmune disease. There is no experimental model for JDM, greatly limiting our understanding of the pathophysiology of the disease process, which, like other autoimmune diseases, is associated with premature cardiovascular compromise and chronic inflammation. Heritable genetic factors, either unique or shared with other autoimmune diseases, predispose the child to develop JDM in concert with environmental influences, such as UVB exposure, season of birth and antecedent infection. Characterization of muscle-specific molecular pathways is severely constrained by the limited amount of involved muscle from the diagnostic biopsy available for research studies. We have documented dysregulation at the mRNA level and aberrant miRNA control in the muscle of untreated children with JDM, as well as active production of cytokines by the muscle fibers themselves, suggesting that they participate in the immune response, and are not only targets of it. A recent discovery showed that mononuclear cells, present in a small amount (5ml) of peripheral blood, can be "reprogrammed" by infecting them with a non-integrating Sendai virus to create " induced pluripotent stem cells" (iPSC). IPSCs share many traits with embryonic stem cells; they can induce the formation of teratomas when transplanted into mice, and give rise to a wide spectrum of differentiated cell types-among them myogenic progenitor cells, which then develop into muscle. In this study we will derive a single cell population of myogenic precursor cells from iPSCs from 3 sets of monozygotic twins discordant for JDM and their matched healthy controls in order to characterize the genetic contribution of muscle to disease pathophysiology. We hypothesize that the differentiated muscle cells derived from iPSCs will recapitulate molecular differences, associated with phenotype that we have previously identified in the transcriptome (miRNAs and mRNAs) of muscle biopsies from untreated children with JDM vs healthy controls. In Specific Aim #1, we will derive and compare iPSCs from three sets of monozygotic twins: one diagnosed with JDM, one unaffected-- and an age, gender, race matched control in order to: a) Verify their pluripotency by teratoma formation in a murine host and, b) Induce the embryoid body cells to form skeletal muscle in vitro. The unaffected twin will serve as a specific genetic and environmental control. Specific Aim # 2 will characterize the transcriptome differences, using RNAseq and miRNA assays, between: 1) the iPSCs from JDM, unaffected twin and healthy matched control as well as 2) their iPSC-derived myogenic cells to determine differences in IPSc and muscle from each donor as well as between donors. The Bioinformatics analysis will normalize the data and identify differentially expressed genes and their pathways in both the iPSCs and their derived muscle cells for each donor. The success of this project will lead to greater understanding of inflammatory myopathy and will open up a new avenue for pathophysiological investigation of JDM and other autoimmune diseases.