Choi Seung-Cheol, Lee Hyunjoo, Choi Ji-Hyun, Kim Jong-Ho, Park Chi-Yeon, Joo Hyung-Joon, Park Jae-Hyoung, Hong Soon-Jun, Yu Cheol-Woong, Lim Do-Sun
Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, Seoul, Korea.
PLoS One. 2015 Jan 28;10(1):e0117410. doi: 10.1371/journal.pone.0117410. eCollection 2015.
Little is known about the mechanisms underlying the effects of Cyclosporin A (CsA) on the fate of stem cells, including cardiomyogenic differentiation. Therefore, we investigated the effects and the molecular mechanisms behind the actions of CsA on cell lineage determination of P19 cells. CsA induced cardiomyocyte-specific differentiation of P19 cells, with the highest efficiency at a concentration of 0.32 μM during embryoid body (EB) formation via activation of the Wnt signaling pathway molecules, Wnt3a, Wnt5a, and Wnt8a, and the cardiac mesoderm markers, Mixl1, Mesp1, and Mesp2. Interestingly, cotreatment of P19 cells with CsA plus dimethyl sulfoxide (DMSO) during EB formation significantly increases cardiac differentiation. In contrast, mRNA expression levels of hematopoietic and endothelial lineage markers, including Flk1 and Er71, were severely reduced in CsA-treated P19 cells. Furthermore, expression of Flk1 protein and the percentage of Flk1+ cells were severely reduced in 0.32 μM CsA-treated P19 cells compared to control cells. CsA significantly modulated mRNA expression levels of the cell cycle molecules, p53 and Cyclins D1, D2, and E2 in P19 cells during EB formation. Moreover, CsA significantly increased cell death and reduced cell number in P19 cells during EB formation. These results demonstrate that CsA induces cardiac differentiation but inhibits hemato-endothelial differentiation via activation of the Wnt signaling pathway, followed by modulation of cell lineage-determining genes in P19 cells during EB formation.
关于环孢菌素A（CsA）对干细胞命运（包括心肌分化）影响的潜在机制，人们了解甚少。因此，我们研究了CsA对P19细胞谱系确定作用背后的影响及分子机制。CsA诱导P19细胞向心肌细胞特异性分化，在胚状体（EB）形成过程中，浓度为0.32μM时效率最高，通过激活Wnt信号通路分子Wnt3a、Wnt5a和Wnt8a以及心脏中胚层标志物Mixl1、Mesp1和Mesp2来实现。有趣的是，在EB形成过程中，用CsA加二甲基亚砜（DMSO）共同处理P19细胞可显著增加心脏分化。相反，在经CsA处理的P19细胞中，包括Flk1和Er71在内的造血和内皮谱系标志物的mRNA表达水平严重降低。此外，与对照细胞相比，在0.32μM CsA处理的P19细胞中，Flk1蛋白的表达和Flk1 +细胞的百分比严重降低。在EB形成过程中，CsA显著调节P19细胞中细胞周期分子p53和细胞周期蛋白D1、D2和E2的mRNA表达水平。此外，在EB形成过程中，CsA显著增加P19细胞的细胞死亡并减少细胞数量。这些结果表明，CsA通过激活Wnt信号通路诱导心脏分化，但抑制造血内皮分化，随后在EB形成过程中调节P19细胞中细胞谱系决定基因。
