Development of iPS cells to study craniometaphyseal dysplasia in humans

描述（申请人提供）：颅干骺端发育不良（CMD）是一种罕见的遗传性颅管骨疾病，始于儿童时期，表现为颅骨肥大和长骨发育不全。在一些患者中，它的终生进展会导致危及生命的后果。到目前为止，除了重复手术，没有其他治疗方法。由于这种疾病的病因复杂，而且用于研究的骨组织很少，因此对CMD的研究还很不足。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）来源，这些干细胞可以分化为参与骨重塑的细胞。这项探索性应用的重点是将来自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。我们将使用由Takahashi等人开发的逆转录病毒载体和方案来生产iPS细胞，并对现有方案进行任何有用的修改。IPS细胞克隆将被彻底表征为正常核型，基因表达谱，表观遗传谱，并将使用畸胎瘤试验测试多能性。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，并采用多种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相似性的成骨细胞。在启动iPS细胞研究之前，我们将使用美国国立卫生研究院批准的人类胚胎干细胞（hESC）来评估和优化现有的将hESC分化为成骨细胞的方案。目前，我们希望将重点放在最初将细胞分化成间充质干细胞样群体，然后在标准条件下将其分化成成骨细胞的方案上。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对CMD（或任何其他颅管疾病）进行广泛研究的模型。公共卫生相关性：该应用程序旨在从皮肤活检中培养人类诱导多能干细胞（IPS），这些细胞能够分化为典型的成熟骨形成细胞（成骨细胞）。本研究的目的是获得罕见骨疾病患者的成骨细胞，以详细研究疾病机制，否则这是不可能的。我们选择从患有颅干骺端发育不良（CMD）（一种使人衰弱的遗传性骨骼疾病）的患者身上制造ips衍生的成骨细胞，因为我们从CMD的小鼠模型中获得了非常有趣的数据，但目前还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.