Changes in gene expression in human skeletal stem cells transduced with constitutively active Gsα correlates with hallmark histopathological changes seen in fibrous dysplastic bone. Changes in gene expression in human skeletal stem cells transduced with constitutively active Gsα correlates with hallmark histopathological changes seen in fibrous dysplastic bone

In order to dissect pathways enchained in skeletal stem/progenitors by Fibrous Dysplasia mutations, we engineered human skeletal stem/progenitors with the mutation and performed transcriptomic analysis. FD mutation profoundly alters the properties of skeletal stem/progenitors by pushing hBMSCs towards formation of disorganized bone with a concomitant lack of fat development. In addition, the mutation created an altered in trans environment that pushed the overall system towards neovascularization, inflammation and osteoclastogenesis. We used microarrays to detail the global programme of gene expressionc of human skeletal progenitors containing the Gsα activating mutation R201C Overall design: Human bone marrow stromal cells (hBMSCs) (derived from bone marrow aspirates) from three independent healthy donors were isolated. Lentiviral vectors (LV-GSαR201C and LV-ctr) were generated, produced and titrated. The LV-vector integrated copy number was calculated by Q-PCR as described and was established as ~1 copy of integrated lentiviral sequence per transduced cell. hBMSCs were transduced with LV-GSαR201C and LV-ctr or mock treated.

为解析纤维发育不良（Fibrous Dysplasia）突变所关联的骨骼干/祖细胞（skeletal stem/progenitors）通路网络，我们对携带该突变的人类骨骼干/祖细胞进行工程化改造，并开展转录组学分析。纤维发育不良突变可通过诱导人类骨髓基质干细胞（human bone marrow stromal cells, hBMSCs）形成紊乱骨组织且伴随脂肪生成缺失，深刻改变骨骼干/祖细胞的特性。此外，该突变还可产生反式调控的异常微环境，推动整体系统朝向新血管生成、炎症反应及破骨细胞生成方向进展。我们通过基因芯片技术，详细表征了携带Gsα激活突变R201C的人类骨骼祖细胞的全局基因表达程序。
整体实验设计：从3名健康供体的骨髓穿刺标本中分离人类骨髓基质干细胞（hBMSCs）。构建、制备并滴定LV-GSαR201C与LV-ctr两种慢病毒载体（Lentiviral vectors, LV）。如前文所述，通过实时定量PCR（Q-PCR）计算慢病毒载体的整合拷贝数，确定每个转导细胞的整合慢病毒序列拷贝数约为1。将hBMSCs分别用LV-GSαR201C、LV-ctr转导，或进行模拟转导处理。