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.
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.
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