背景:胚胎干细胞(ESC)作为一个重要的离体模型,代表来自胚泡期胚胎的内细胞团的上胚细胞。ESC表现出独特的自我更新效能组合,无限扩散,和多能性。后者通过分离的细胞自发分化成多个细胞谱系的能力来证明。代表三个主要的胚胎胚层。多个监管网络指导ESC,指导他们的自我更新和谱系特异性分化。细胞凋亡,或程序性细胞死亡,作为涉及雕刻和形成确保适当胚胎发育的各种器官和结构的关键事件而出现。然而,分化和凋亡之间的动态相互作用的分子机制仍然知之甚少。
目的:探讨细胞凋亡对胚胎干细胞早期分化为心肌细胞的调控作用。使用小鼠ESC(mESC)模型-mESC-B细胞淋巴瘤2(BCL-2),mESC-PIM-2和mESC-金属硫蛋白-1(MET-1)-分别过表达抗凋亡基因Bcl-2,Pim-2和Met-1。
方法:mESC-T2(野生型),mESC-BCL-2、mESC-PIM-2和mESC-MET-1已用于评估增强的凋亡信号对心脏分化的影响。采用悬滴法产生胚状体(EBs)并诱导mESCs终末分化。与野生型相比,在每种条件下测量产生的EB的大小。在功能层面,通过计算与对照相比的经操纵的mESC中搏动心肌细胞的数量来测量心脏分化的百分比。在分子水平上,定量逆转录聚合酶链反应用于评估三种心脏标志物的mRNA表达:肌钙蛋白T,GATA4和NKX2.5。此外,肌钙蛋白T蛋白的表达通过免疫荧光和蛋白质印迹分析进行评估。
结果:我们的发现表明,Bcl-2、Pim-2和Met-1基因的上调导致来自被操纵的mESCs的EBs大小减少,与它们的野生型对应物相比。此外,观察到分化细胞中跳动的心肌细胞计数减少。此外,三种心脏标志物-肌钙蛋白T的mRNA表达,与对照细胞系相比,GATA4和NKX2.5-在过表达三个抗凋亡基因的mESC中减少。此外,抗凋亡基因的过度表达导致肌钙蛋白T蛋白表达减少.
结论:我们的发现表明Bcl-2、Pim-2和Met-1基因的上调改变了心脏分化,提供对细胞凋亡和ESC命运决定之间复杂相互作用的洞察。
BACKGROUND: Embryonic stem cells (ESCs) serve as a crucial ex vivo model, representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos. ESCs exhibit a unique combination of self-renewal potency, unlimited proliferation, and pluripotency. The latter is evident by the ability of the isolated cells to differentiate spontaneously into multiple cell lineages, representing the three primary embryonic germ layers. Multiple regulatory networks guide ESCs, directing their self-renewal and lineage-specific differentiation. Apoptosis, or programmed cell death, emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development. However, the molecular mechanisms underlying the dynamic interplay between differentiation and apoptosis remain poorly understood.
OBJECTIVE: To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells, using mouse ESC (mESC) models - mESC-B-cell lymphoma 2 (BCL-2), mESC-PIM-2, and mESC-metallothionein-1 (MET-1) - which overexpress the anti-apoptotic genes Bcl-2, Pim-2, and Met-1, respectively.
METHODS: mESC-T2 (wild-type), mESC-BCL-2, mESC-PIM-2, and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation. The hanging drop method was adopted to generate embryoid bodies (EBs) and induce terminal differentiation of mESCs. The size of the generated EBs was measured in each condition compared to the wild type. At the functional level, the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control. At the molecular level, quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers: Troponin T, GATA4, and NKX2.5. Additionally, troponin T protein expression was evaluated through immunofluorescence and western blot assays.
RESULTS: Our findings showed that the upregulation of Bcl-2, Pim-2, and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs, in comparison with their wild-type counterpart. Additionally, a decrease in the count of beating cardiomyocytes among differentiated cells was observed. Furthermore, the mRNA expression of three cardiac markers - troponin T, GATA4, and NKX2.5 - was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line. Moreover, the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression.
CONCLUSIONS: Our findings revealed that the upregulation of Bcl-2, Pim-2, and Met-1 genes altered cardiac differentiation, providing insight into the intricate interplay between apoptosis and ESC fate determination.