PhyMedExp, INSERM, University of Montpellier, CNRS, 34000 Montpellier, France.
Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic.
Int J Mol Sci. 2021 Jan 11;22(2):662. doi: 10.3390/ijms22020662.
Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O or LOE) and high oxygen exposure (21% O or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca release properties revealing a better SR Ca handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.
已经建立了许多心脏分化方案，主要集中在特定的生长因子对人多能干细胞（hPSC）分化效率的影响上。然而，获得网络中心肌细胞的最佳环境因素仍不清楚。中胚层的分化已知会被低氧暴露所增强。在这里，我们假设低氧暴露会增强心肌细胞的分子和功能成熟度。我们旨在比较低氧（5% O 或 LOE）和高氧（21% O 或 HOE）暴露对 hPSC 在二维和三维分化方案中分化为心肌细胞的分子和功能后果。通过多种细胞系，通过 2D（单层）和 3D（胚状体）方案来分化 hPSC-CMs。评估了心肌标志物的表达和细胞形态。评估了线粒体定位和代谢特性。还监测了细胞内 Ca 处理和收缩特性。二维心脏单层仅能在 HOE 中分化。LOE 中的 3D 心脏球体含有 hPSC-CMs，进一步表现出心肌标志物、肥大、更稳定的 SR Ca 释放特性，表明 SR Ca 处理更好，收缩力增强。还观察到线粒体的分布保持不变，线粒体呼吸链复合物的耗氧量相似。我们的结果提供了证据表明，LOE 对具有 hPSC-CMs 的 3D 心脏球体具有适度的益处，表现出进一步的成熟。相比之下，二维心脏单层严格需要 HOE。
