Wnt-GSK3/-Catenin Regulates the Differentiation of Dental Pulp Stem Cells into Bladder Smooth Muscle Cells

Smooth muscle cell- (SMC-) based tissue engineering provides a promising therapeutic strategy for SMC-related disorders. It has been demonstrated that human dental pulp stem cells (DPSCs) possess the potential to differentiate into mature bladder SMCs by induction with condition medium (CM) from bladder SMC culture, in combination with the transforming growth factor-1 (TGF-1). However, the molecular mechanism of SMC differentiation from DPSCs has not been fully uncovered. The canonical Wnt signaling (also known as Wnt/-catenin) pathway plays an essential role in stem cell fate decision. The aim of this study is to explore the regulation via GSK3 and associated downstream effectors for SMC differentiation from DPSCs. We characterized one of our DPSC clones with the best proliferation and differentiation abilities. This stem cell clone has shown the capacity to generate a smooth muscle layer-like phenotype after an extended differentiation duration using the SMC induction protocol we established before. We further found that Wnt-GSK3/-catenin signaling is involved in the process of SMC differentiation from DPSCs, as well as a serial of growth factors, including TGF-1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor-homodimer polypeptide of B chain (BB) (PDGF-BB), and vascular endothelial growth factor (VEGF). Pharmacological inhibition on the canonical Wnt-GSK3/-catenin pathway significantly downregulated GSK3 phosphorylation and -catenin activation, which in consequence reduced the augmented expression of the growth factors (including TGF-1, HGF, PDGF-BB, and VEGF) as well as SMC markers (especially myosin) at a late stage of SMC differentiation. These results suggest that the canonical Wnt-GSK3/-catenin pathway contributes to DPSC differentiation into mature SMCs through the coordination of different growth factors.

基于平滑肌细胞（SMC）的组织工程为SMC相关疾病提供了一种有前景的治疗策略。已证实人牙髓干细胞（DPSCs）在膀胱平滑肌细胞培养的条件培养基（CM）以及转化生长因子 -β1（TGF -β1）的诱导下，具有分化为成熟膀胱平滑肌细胞的潜能。然而，DPSCs向平滑肌细胞分化的分子机制尚未完全阐明。经典Wnt信号通路（也称为Wnt/β -连环蛋白通路）在干细胞命运决定中起着关键作用。本研究的目的是探索通过糖原合成酶激酶3（GSK3）及其相关下游效应因子对DPSCs向平滑肌细胞分化的调控作用。我们对增殖和分化能力最佳的一个DPSC克隆进行了特性分析。使用我们之前建立的平滑肌细胞诱导方案，这个干细胞克隆在延长分化时间后显示出产生平滑肌层样表型的能力。我们进一步发现，Wnt - GSK3/β -连环蛋白信号通路参与了DPSCs向平滑肌细胞分化的过程，还有一系列生长因子也参与其中，包括TGF -β1、碱性成纤维细胞生长因子（bFGF）、表皮生长因子（EGF）、肝细胞生长因子（HGF）、血小板衍生生长因子 - B链同型二聚体多肽（BB）（PDGF - BB）和血管内皮生长因子（VEGF）。对经典Wnt - GSK3/β -连环蛋白通路的药理学抑制显著下调了GSK3的磷酸化和β -连环蛋白的激活，从而降低了平滑肌细胞分化后期生长因子（包括TGF -β1、HGF、PDGF - BB和VEGF）以及平滑肌细胞标志物（特别是肌球蛋白）的增强表达。这些结果表明，经典Wnt - GSK3/β -连环蛋白通路通过不同生长因子的协同作用促进DPSC分化为成熟的平滑肌细胞。