BMP4 Signaling Plays a Critical Roles in Self-renewal of R2i Mouse Embryonic Stem Cells. BMP4 Signaling Plays a Critical Roles in Self-renewal of R2i Mouse Embryonic Stem Cells

Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state under R2i culture conditions that inhibit the TGF-β and ERK signaling pathways. BMP4 is another member of the TGF-β family that plays a crucial role in maintaining the pluripotency state of mESCs. It has been reported that inhibition of BMP4 caused the death of R2i-grown cells. In this study, we used the loss-of-function approach to investigate the role of BMP4 signaling in mESC self-renewal. Inhibition of this pathway with Noggin and dorsomorphin, two bone morphogenetic protein (BMP) antagonists, elicited a quick death of the R2i-grown cells. We showed that the canonical pathway of BMP4 (BMP/SMAD) was dispensable for self-renewal and maintaining pluripotency of these cells. Transcriptome analysis of the BMPi-treated cells revealed that the p53 signaling and two adhesion (AD) and apoptotic mitochondrial change (MT) pathways could be involved in the cell death of the BMPi-treated cells. According to our results, inhibition of BMP4 signaling caused a decrease in cell adhesion and ECM detachment, which triggered anoikis in the R2i-grown cells. Altogether, these findings demonstrate that endogenous BMP signaling is required for survival of mESCs under the R2i condition. Overall design: Examination of BMP4 signaling role in mESC self-renewal

小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）可在抑制转化生长因子-β（transforming growth factor-β, TGF-β）与细胞外调节蛋白激酶（extracellular regulated protein kinases, ERK）信号通路的R2i培养条件下维持其多能状态。骨形态发生蛋白4（bone morphogenetic protein 4, BMP4）属于TGF-β家族成员，在维持mESCs多能状态的过程中发挥关键调控作用。已有研究证实，抑制BMP4会导致R2i培养体系中的细胞死亡。本研究采用功能缺失策略，探究BMP4信号通路在mESC自我更新中的作用。使用两种骨形态发生蛋白拮抗剂——Noggin与dorsomorphin——抑制该通路时，可快速引发R2i培养体系下的细胞死亡。本研究结果显示，BMP4的经典通路（BMP/SMAD）对于这些细胞的自我更新与多能状态维持并非必需。对BMP抑制剂处理后的细胞进行转录组分析后发现，p53信号通路、两类黏附（AD）通路以及凋亡线粒体改变（MT）通路，可能参与了BMP抑制剂处理引发的细胞死亡过程。根据本研究结果，抑制BMP4信号通路会导致细胞黏附能力下降与细胞外基质（extracellular matrix, ECM）脱离，进而触发R2i培养体系下细胞的失巢凋亡。综上，本研究证实，在R2i培养条件下，内源性BMP信号通路是mESCs存活所必需的。实验设计概要：探究BMP4信号通路在mESC自我更新中的作用