Le Minh Nguyen Tuyet, Takahi Mika, Maruyama Kenshiro, Kurisaki Akira, Ohnuma Kiyoshi
Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188, Japan.
Department of Science of Technology Innovation, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188, Japan.
In Vitro Cell Dev Biol Anim. 2018 Aug;54(7):513-522. doi: 10.1007/s11626-018-0276-0. Epub 2018 Jul 2.
A high density of human-induced pluripotent stem cells (hiPSCs) improves the efficiency of cardiac differentiation, suggesting the existence of indispensable cell-cell interaction signals. The complexity of interactions among cells at high density hinders the understanding of the roles of cell signals. In this study, we determined the minimum cell density that can initiate differentiation to facilitate cell-cell interaction studies. First, we co-induced cardiac differentiation in the presence of the glycogen synthase kinase-3β inhibitor CHIR99021 and activin A at various cell densities. At an initial low density, cells died within a few days in RPMI-based medium. We then investigated the culture conditions required to maintain cell viability. We used a basal medium excluding important components for the maintenance of hiPSC pluripotency, including activin A, basic fibroblast growth factor, and insulin. Supplementation of the basal medium with Rho-associated protein kinase inhibitor and insulin improved cell viability. Interestingly, addition of basic fibroblast growth factor enabled the expression of cardiac markers at the mRNA level but not the protein level. After further modification of the culture conditions, 10% of the cells expressed the cardiac troponin T protein, which is associated with cell contraction. The novel protocol for cardiac differentiation at an initial low cell density can also be used to evaluate high cell density conditions. The findings will facilitate the identification of cell signals required for cardiomyocyte formation.
高密度的人诱导多能干细胞（hiPSC）可提高心脏分化效率，这表明存在不可或缺的细胞间相互作用信号。高密度下细胞间相互作用的复杂性阻碍了对细胞信号作用的理解。在本研究中，我们确定了能够启动分化以促进细胞间相互作用研究的最小细胞密度。首先，我们在糖原合酶激酶-3β抑制剂CHIR99021和激活素A存在的情况下，于不同细胞密度下共同诱导心脏分化。在初始低密度时，细胞在基于RPMI的培养基中几天内就死亡了。然后，我们研究了维持细胞活力所需的培养条件。我们使用了一种基础培养基，该培养基不含维持hiPSC多能性的重要成分，包括激活素A、碱性成纤维细胞生长因子和胰岛素。向基础培养基中添加Rho相关蛋白激酶抑制剂和胰岛素可提高细胞活力。有趣的是，添加碱性成纤维细胞生长因子能使心脏标志物在mRNA水平表达，但在蛋白质水平却不能。在对培养条件进一步优化后，10%的细胞表达了与细胞收缩相关的心肌肌钙蛋白T蛋白。这种在初始低细胞密度下进行心脏分化的新方案也可用于评估高细胞密度条件。这些发现将有助于识别心肌细胞形成所需的细胞信号。
