Transcriptional Analysis of Fracture Healing and the Induction of Embryonic Stem Cell�CRelated Genes

Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and ��350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture healing. They show that neurogenesis as well as vasculogenesis are predominant components of skeletal tissue formation and suggest common pathways are shared between post-natal stem cells and those seen in ESCs.

骨折是人类最常见的创伤性疾病之一。骨折愈合是一种独特的、可通过时序界定的出生后生物学过程，为研究调控软骨内骨骼组织形成的多种分子事件间的复杂相互作用提供了理想研究模型。鉴于骨折愈合具有再生修复特性，学界推测该过程会招募大量成体干细胞，并参与构成此过程所需的多种细胞谱系。本研究采用贝叶斯建模（Bayesian modeling）方法，构建了骨折愈合过程中转录组（transcriptome）的时序表达谱。研究鉴定了与多种生物学、代谢及调控通路相关的各类基因本体（ontologies）间的时序关联，并将其与骨骼发生、血管生成及神经发生相关的发育过程进行关联分析。本研究还将所有表达的骨形态发生蛋白（Bone Morphogenetic Proteins, BMPs）、Wnt信号通路分子（Wnts）、成纤维细胞生长因子（Fibroblast Growth Factors, FGFs）及其受体的整体表达情况，与骨折愈合过程中同步表达的转录因子亚群进行了关联分析。此外，本研究明确了193个已知与人类遗传性骨骼疾病相关的基因中，174个基因在骨折愈合过程中的时序表达模式。为鉴定骨折愈合招募的干细胞与其他类型干细胞共有的调控特征，本研究将骨折愈合的转录组数据与胚胎干细胞（embryonic stem cells, ESCs）及间充质干细胞（mesenchymal stem cells, MSCs）的转录组数据进行了比对检索。约300个在ESCs中优先表达的已知基因，以及约350个在MSCs中优先表达的已知基因，在骨折愈合过程中呈现诱导上调表达。Nanog作为维持ESCs干性的核心表观调控因子之一，被证实与多种骨修复过程及MSCs分化密切相关。综上，本研究首次对骨折愈合过程中发生的软骨内骨形成过程的转录组进行了时序分析。研究结果显示，神经发生与血管生成是骨骼组织形成的核心组成部分，同时提示成体干细胞与胚胎干细胞共享部分调控通路。