College of Pharmacy and Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 21999, Korea.
Department of Marine Bio and Medical Sciences, Hanseo University, Seonsan-si 31962, Korea.
Int J Mol Sci. 2021 Sep 29;22(19):10532. doi: 10.3390/ijms221910532.
Pluripotent adult stem cells have potential applications in cell therapy and tissue engineering. Urine-derived stem cells (UDSCs) differentiate into various cell types. Here, we attempted to differentiate human UDSCs (hUDSCs) into smooth muscle cells (SMCs) using transforming growth factor-beta 1 (TGF-β1) and/or PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Both quantitative polymerase chain reaction (qPCR) and Western blot analysis showed that the expression of messenger ribonucleic acid (mRNA) and proteins for alpha-smooth muscle actin (α-SMA), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC), which are specific markers for SMCs, increased on day 9 after differentiation and again on day 14. The differentiated cells from human UDSCs (hUDSCs) with a combination of TGF-β1 and PD98059 showed the highest expression of SMC marker proteins. Immunocytochemical staining performed to assess the molecular expression revealed CNN and α-SMA colocalizing in the cytoplasm. The cells that differentiated from hUDSCs with a combination of TGF-β1 and PD98059 showed the strongest expression for CNN1, α-SMA, and SM-MHC. Functional testing of the differentiated cells revealed a stronger contractile capacity for the cells differentiated with a combination of PD98059 and TGF-β1 than those differentiated with a single factor. These results suggest the combination of PD98059 and TGF-β1 to be a more effective differentiation method and that differentiated SMCs could be used for restoring the functions of the sphincter muscle or bladder.
多能成体干细胞在细胞治疗和组织工程中有潜在的应用。尿源干细胞（UDSCs）可分化为多种细胞类型。在这里，我们试图使用转化生长因子-β1（TGF-β1）和/或 PD98059（一种细胞外信号调节激酶（ERK）抑制剂）将人尿源干细胞（hUDSCs）分化为平滑肌细胞（SMCs）。实时定量聚合酶链反应（qPCR）和 Western blot 分析均显示，在分化后第 9 天和第 14 天，α-平滑肌肌动蛋白（α-SMA）、钙调蛋白（CNN1）和平滑肌肌球蛋白重链（SM-MHC）信使核糖核酸（mRNA）和蛋白的表达增加，这些蛋白是 SMC 的特异性标志物。TGF-β1 和 PD98059 联合作用下分化的 hUDSCs 中的分化细胞显示出最高的 SMC 标志物蛋白表达。进行免疫细胞化学染色以评估分子表达，结果显示 CNN 和 α-SMA 在细胞质中共定位。TGF-β1 和 PD98059 联合作用下分化的 hUDSCs 分化的细胞表达最强的 CNN1、α-SMA 和 SM-MHC。对分化细胞的功能测试表明，与单一因素分化的细胞相比，PD98059 和 TGF-β1 联合作用下分化的细胞具有更强的收缩能力。这些结果表明 PD98059 和 TGF-β1 的联合使用是一种更有效的分化方法，分化的 SMC 可用于恢复括约肌或膀胱的功能。
