School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, PR China.
Biochemistry (Mosc). 2015 Mar;80(3):379-89. doi: 10.1134/S000629791503013X.
The roles of sugar chains such as heparan sulfate (HS) in stem cell self-renewal and differentiation are poorly understood. HS is a sugar chain with linear sulfated polyanionic disaccharide repeating structures that interact with many proteins, including structural proteins in the extracellular matrix and growth factors and their receptors. Thus, unraveling the role of HS in stem cell self-renewal and differentiation could provide new insights and technical routes in clinical stem cell applications. Here, we purified rat bone marrow mesenchymal stromal cells (BMMSCs) by density gradient centrifugation, analyzed mesenchymal stromal cell surface stemness marker expression by flow cytometry, and identified the sulfotransferases responsible for sulfation ester modification of HS. An osteogenic differentiation model was established by chemical induction reagents and confirmed via alkaline phosphatase (ALP) activity detection and the expression of the osteogenic differentiation markers Runx2 and Ocn. The expression profiles of HS sulfotransferases in rat BMMSCs before and after osteogenic induction were detected by RT-PCR and Western blot. Cell spheroids were formed in both control and osteogenic culture systems when BMMSCs were grown to high confluence. We determined that this type of cell spheroid was a highly calcified nodule by histochemical staining. Among all the sulfotransferases examined, heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) mRNA and protein were upregulated in these calcified cell spheroids. HS6ST3 knockdown BMMSCs were established with RNA interference, and they had significantly lower ALP activity and decreased expression of the osteogenic differentiation markers Runx2 and Ocn. These findings suggest that HS6ST3 is involved in BMMSC differentiation, and new glycotherapeutic-based technologies could be developed in the future.
糖链（如硫酸乙酰肝素 (HS)）在干细胞自我更新和分化中的作用还知之甚少。HS 是一种具有线性硫酸化聚阴离子二糖重复结构的糖链，与许多蛋白质相互作用，包括细胞外基质中的结构蛋白、生长因子及其受体。因此，阐明 HS 在干细胞自我更新和分化中的作用可能为临床干细胞应用提供新的见解和技术途径。在这里，我们通过密度梯度离心法纯化大鼠骨髓间充质基质细胞（BMMSCs），通过流式细胞术分析间充质基质细胞表面干性标志物的表达，并鉴定负责 HS 硫酸酯化修饰的硫酸转移酶。通过化学诱导剂建立成骨分化模型，并通过碱性磷酸酶（ALP）活性检测和骨向分化标志物 Runx2 和 Ocn 的表达进行验证。通过 RT-PCR 和 Western blot 检测大鼠 BMMSCs 成骨诱导前后 HS 硫酸转移酶的表达谱。当 BMMSCs 生长到高汇合度时，在对照和成骨培养系统中均形成细胞球。我们通过组织化学染色确定这种类型的细胞球是一种高度钙化的结节。在所检查的所有硫酸转移酶中，肝素硫酸 6-O-硫酸转移酶 3（HS6ST3）mRNA 和蛋白在这些钙化细胞球中上调。用 RNA 干扰建立了 HS6ST3 敲低的 BMMSCs，其 ALP 活性显著降低，骨向分化标志物 Runx2 和 Ocn 的表达降低。这些发现表明 HS6ST3 参与 BMMSC 分化，未来可能开发新的糖治疗技术。
