AhR alters essential transcriptional regulators driving osteogenic differentiation of multipotent mesenchymal stem cells. AhR alters essential transcriptional regulators driving osteogenic differentiation of multipotent mesenchymal stem cells

Differentiation of multipotent mesenchymal stem cells into bone-forming osteoblasts requires strict coordination of transcriptional pathways. Aryl hydrocarbon receptor (AhR) ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been shown to alter osteoblast differentiation in vitro and bone formation in multiple developmental in vivo models. The goal of the present study was to establish a global transcriptomic landscape during early, intermediate, and apical stages of osteogenic differentiation in vitro in response to TCDD exposure. Human bone-derived mesenchymal stem cells (hBMSC) were cultured in growth media (GM), osteogenic differentiation media (ODM), or osteogenic differentiation media containing 10 nM TCDD (ODM+TCDD), thus enabling a comparison of the transcriptomic profiles of undifferentiated, differentiated, and differentiated -TCDD-exposed hBMSCs, respectively. In this test system, exposure to TCDD attenuated differentiation of hBMSCs into osteoblasts as evidenced by reduced alkaline phosphatase activity and mineralization. At various timepoints, we observed altered expression of genes that play a role in the Wnt, FGF, BMP/TGF-β developmental pathways, as well as pathways related to extracellular matrix organization and deposition. Reconstruction of gene regulatory networks with the iDREM analysis revealed modulation of transcription factors (TF) including POLR3G, NR4A1, RDBP, GTF2B, POU2F2 and ZEB1, which may putatively influence osteoblast differentiation and the requisite deposition and mineralization of bone extracellular matrix. We demonstrate that the combination of RNA-Seq data in conjunction with the iDREM regulatory model, captures the transcriptional dynamics underlying mesenchymal stem cell differentiation under different conditions in vitro. Model predictions are consistent with existing knowledge and provides a new tool to identify novel pathways and transcription factors that may facilitate a better understanding of the osteoblast differentiation process, perturbation by exogenous agents, and potential intervention strategies targeting those specific pathways. Overall design: TCDD alters essential transcriptional regulators of osteogenic differentiation in multipotent mesenchymal stem cells

多能间充质干细胞（multipotent mesenchymal stem cells）向成骨细胞（bone-forming osteoblasts）分化需要转录通路的严格协调。芳基烃受体（aryl hydrocarbon receptor, AhR）配体，例如2,3,7,8-四氯二苯并对二噁英（2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD），已被证实可在体外改变成骨细胞分化，并在多种体内发育模型中影响骨形成。本研究的目标是构建体外成骨分化早期、中期及终末阶段经TCDD暴露后的全局转录组图谱。人类骨源性间充质干细胞（human bone-derived mesenchymal stem cells, hBMSC）分别在生长培养基（growth media, GM）、成骨分化培养基（osteogenic differentiation media, ODM），或添加10 nM TCDD的成骨分化培养基（ODM+TCDD）中培养，由此可分别比较未分化、已分化及经TCDD暴露的已分化hBMSCs的转录组特征。在该实验体系中，TCDD暴露可减弱hBMSCs向成骨细胞的分化，这一点可通过碱性磷酸酶活性降低与矿化水平下降得到证实。在不同时间点，我们观察到参与Wnt、FGF、BMP/TGF-β发育通路，以及与细胞外基质组织和沉积相关通路的基因表达发生改变。通过iDREM分析构建基因调控网络后发现，包括POLR3G、NR4A1、RDBP、GTF2B、POU2F2及ZEB1在内的转录因子（transcription factors, TF）的调控发生变化，这些因子可能潜在影响成骨细胞分化以及骨细胞外基质必要的沉积与矿化。本研究表明，将RNA测序（RNA-Seq）数据与iDREM调控模型相结合，可捕捉体外不同条件下间充质干细胞分化的转录动态。模型预测结果与现有认知相符，并为识别新的通路和转录因子提供了新工具，有助于更好地理解成骨细胞分化过程、外源性物质的扰动作用，以及针对这些特定通路的潜在干预策略。整体实验设计：TCDD可改变多能间充质干细胞中成骨分化的关键转录调控因子。