Development of iPS cells to study craniometaphyseal dysplasia in humans

描述（由申请人提供）：颅骨骺发育不良（CMD）是一种罕见的遗传性颅管状骨疾病，始于儿童时期，伴有颅骨肥大和长骨发育不良。其终身进展导致一些患者危及生命的后果。除了重复手术外，目前还没有其他治疗方法。CMD是严重不足的研究，因为这种疾病的病因是复杂的，骨组织的研究很少。因此，我们建议从CMD患者和对照个体中创建诱导多能干细胞（iPS）的来源，其可以分化为参与骨重建的细胞。该探索性应用的重点是将CMD患者和正常对照个体的皮肤成纤维细胞重编程为iPS细胞。我们将使用Takahashi等人开发的逆转录病毒载体和方案来生产iPS细胞，并对可用的方案进行任何有用的修改。将对IPS细胞克隆的正常核型、基因表达谱、表观遗传谱进行全面表征，并使用畸胎瘤测定法检测多能性。我们建议在培养中将合适的对照和CMD iPS克隆分化为成骨细胞，并使用广泛的方法详细分析其分化和骨基质沉积的潜力。我们的目标是获得与原代成骨细胞具有最大可能相似性的成骨细胞。在开始iPS细胞的研究之前，我们将使用NIH批准的人胚胎干细胞（hESC）来评估和优化现有的将hESC分化为成骨细胞的方案。目前，我们希望专注于最初将细胞分化为间充质干细胞样群体，然后使用标准条件将其分化为成骨细胞的方案。在时间和资源允许的情况下，我们将对来自CMD患者和对照个体的iPS细胞衍生的成骨细胞进行初步表型比较。这个模型将是第一个允许用人类细胞广泛研究CMD（或任何其他颅管疾病）的模型。公共卫生相关性：本申请旨在从皮肤活检中开发人类诱导性多能干细胞（IPS），这些细胞能够分化为典型的成熟骨形成细胞（成骨细胞）。本研究的目的是从罕见骨疾病患者中获得成骨细胞，用于疾病机制的详细研究，否则这是不可能的。我们选择从颅骺发育不良（CMD）患者中创建IPS衍生的成骨细胞，CMD是一种使人衰弱的遗传性骨骼疾病，因为我们从CMD小鼠模型中获得了非常有趣的数据，但目前不可能将我们的发现转化为人类系统。

DESCRIPTION (provided by applicant): Craniometaphyseal dysplasia (CMD) is a rare genetic craniotubular bone disorder which begins in childhood with cranial hyperostosis and long bone hypoostosis. Its lifelong progression leads to life-threatening consequences in some patients. There is no treatment other than repetitive surgery as of yet. CMD is heavily under-studied because the pathoetiology of this disorder is complex and bone tissue for research is rarely available. Therefore, we propose to create a source of induced pluripotent stem cells (iPS) from CMD patients and control individuals, which can be differentiated into cells involved in bone remodeling. This exploratory application focuses on the reprograming of skin fibroblasts from CMD patients and normal control individuals into iPS cells. We will use retrovirus vectors and protocols developed by Takahashi et al. to produce iPS cells and apply any useful modifications to the protocol that become available. IPS cell clones will be thoroughly characterized for normal karyotype, gene expression profile, epigenetic profile and will be tested for pluripotency using teratoma assays. We propose to differentiate suitable control and CMD iPS clones into osteoblasts in culture and analyze their potential for differentiation and bone matrix deposition in detail with a broad range of methods. Our goal is to obtain osteoblasts with properties that have the greatest possible similarity to primary osteoblasts. Before initiating studies with iPS cells, we will use NIH-approved human embryonic stem cells (hESC) to evaluate and optimize existing protocols for differentiating hESC into osteoblasts. Currently we expect to concentrate on protocols that initially differentiate the cells into mesenchymal stem cell-like populations, and then differentiate them into osteoblasts using standard conditions. As time and resources allow, we will perform initial phenotypic comparisons of iPS cell-derived osteoblasts from CMD patients and control individuals. This model will be the first to allow extensive studies of CMD (or any other craniotubular disorder) with human cells. PUBLIC HEALTH RELEVANCE: This application is designed to develop human inducible pluripotent stem (IPS) cells from skin biopsies, which are capable to differentiate into typical mature bone forming cells (osteoblasts). The goal of this investigation is to obtain osteoblasts from patients with rare bone disorders for detailed studies of disease mechanisms, which would otherwise not be possible. We chose to create IPS-derived osteoblasts from patients with craniometaphyseal dysplasia (CMD), a debilitating genetic bone disorder, because we have very interesting data from a mouse model for CMD but it is currently impossible to translate our findings to the human system.