Osteogenic transdifferentiation of primary human fibroblasts to osteoblast-like cells with human platelet lysate

Inherited bone disorders account for about 10% of documented Mendelian disorders and are associated with high financial burden. Their study requires osteoblasts which play a critical role in regulating the development and maintenance of bone tissue. However, bone tissue is not always available from patients. We developed a highly efficient platelet lysate-based approach to directly transdifferentiate skin-derived human fibroblasts to osteoblast-like cells. We extensively characterized our in vitro model by examining the expression of osteoblast-specific markers during the transdifferentiation process both at the mRNA and protein level. The transdifferentiated osteoblast-like cells showed significantly increased expression of a panel of osteogenic markers. Mineral deposition and ALP activity were also shown, confirming their osteogenic properties. RNA-seq analysis allowed the global study of changes in the transcriptome of the transdifferentiated cells. The transdifferentiated cells clustered separately from the fibroblasts with regard to the significantly upregulated genes indicating a distinct transcriptome profile; transdifferentiated osteoblasts also showed significant enrichment in gene expression related to skeletal development and bone mineralization. Our presented in vitro model may potentially contribute to the prospect of studying osteoblast-dependent disorders in patient-derived cells. Comparative gene expression profiling analysis of RNA-seq data for human fibroblasts undergone osteogenic transdifferentiation

遗传性骨病（Inherited bone disorders）约占已报道孟德尔遗传病（Mendelian disorders）的10%，且与高昂的经济负担密切相关。此类疾病的研究离不开成骨细胞（osteoblasts）——成骨细胞在调控骨组织的发育与稳态维持中发挥关键作用。然而，临床患者往往难以提供骨组织样本。本研究开发了一种基于血小板裂解液的高效方法，可直接将皮肤来源的人成纤维细胞（fibroblasts）转分化为类成骨细胞。我们通过在mRNA与蛋白质两个层面检测转分化过程中成骨细胞特异性标志物的表达水平，对该体外模型进行了全面的表征分析。转分化得到的类成骨细胞的一系列成骨标志物表达量均显著上调。该细胞可形成矿化沉积并表现出碱性磷酸酶（alkaline phosphatase, ALP）活性，证实了其成骨特性。RNA测序（RNA-seq）分析可全局解析转分化细胞的转录组变化。在显著上调基因的表达层面，转分化细胞与成纤维细胞的聚类结果相互分离，提示二者具有截然不同的转录组特征；转分化类成骨细胞的基因表达还显著富集于骨骼发育与骨矿化相关的通路。本研究构建的体外模型有望为利用患者来源细胞研究成骨细胞依赖性疾病提供助力。针对经历成骨转分化的人成纤维细胞的RNA-seq数据所开展的比较基因表达谱分析。