背景:目前的方案在体外产生高纯度的人诱导多能干细胞衍生的心肌细胞(hiPSC-CM),其概括了成熟体内心肌细胞的特征。然而,当将hiPSC-CM注射到大型动物模型中时,存在心律失常的风险.因此,了解hiPSC-CM成熟机制对临床翻译至关重要。叉头盒(FOX)转录因子通过FOXO和FOXM1之间的平衡调节出生后心肌细胞的成熟。我们以前也证明了p53激活增强hiPSC-CM成熟。这里,我们研究了在三维悬浮培养中p53激活是否调节FOXO/FOXM1平衡以促进hiPSC-CM成熟。
结果:用Nutlin-3a(p53激活剂,10μM),LOM612(FOXO重新定位器,5μM),AS1842856(FOXO抑制剂,1μM),或RCM-1(FOXM1抑制剂,1μM),从殴打开始2天后开始,以二甲基亚砜(0.2%载体)作为对照。P53激活促进hiPSC-CM代谢和电生理成熟,同时FOXO上调和FOXM1下调,对于所有测定,n=每组3至6。FOXO抑制显著降低心脏特异性标志物如TNNT2的表达。相比之下,FOXO激活或FOXM1抑制促进成熟特征,如增加的收缩性,耗氧量,和电压峰值最大上冲程速度,对于所有测定,n=每组3至6。Further,通过与二甲基亚砜相比,n=2LOM612处理细胞的单细胞RNA测序,LOM612介导的FOXO激活促进心脏成熟途径的表达。
结论:我们表明,在三维hiPSC-CM成熟过程中,p53激活促进FOXO并抑制FOXM1。这些结果扩展了我们对临床相关的3维培养系统中hiPSC-CM成熟机制的理解。
BACKGROUND: Current protocols generate highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate characteristics of mature in vivo cardiomyocytes. Yet, a risk of arrhythmias exists when hiPSC-CMs are injected into large animal models. Thus, understanding hiPSC-CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC-CM maturation. Here, we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC-CM maturation in 3-dimensional suspension culture.
RESULTS: Three-dimensional cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator, 10 μM), LOM612 (FOXO relocator, 5 μM), AS1842856 (FOXO inhibitor, 1 μM), or RCM-1 (FOXM1 inhibitor, 1 μM), starting 2 days after onset of beating, with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC-CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation, in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility, oxygen consumption, and voltage peak maximum upstroke velocity, in n=3 to 6 per group for all assays. Further, by single-cell RNA sequencing of n=2 LOM612-treated cells compared with dimethyl sulfoxide, LOM612-mediated FOXO activation promoted expression of cardiac maturational pathways.
CONCLUSIONS: We show that p53 activation promotes FOXO and suppresses FOXM1 during 3-dimensional hiPSC-CM maturation. These results expand our understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3-dimensional culture system.