Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, Osaka 565-8565, Japan.
Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, Osaka 565-8565, Japan.
Acta Biomater. 2018 Jan;65:44-52. doi: 10.1016/j.actbio.2017.10.032. Epub 2017 Oct 21.
Stem cell-based myocardial regeneration therapies have emerged as alternative strategies to heart transplantation for serious heart diseases, but autologous beating mature cardiomyocytes are not available. Here we investigated the effect of culture substrates on the cardiomyocyte differentiation of induced pluripotent stem cells (iPSs) in vitro by separately evaluating the following continuous three steps: (1) cardiac marker gene expression, (2) contractile gene expression and self-beating, and (3) beating duration. To this end, we used iPS cells to study the cardiac differentiation, and neonatal rat cardiomyocytes (NCMs) to study beating behavior. These cells were cultured on substrates with different natures, i.e., an elastic substrate (Es) with the modulus of 9, 20, or 180 kPa, and hard tissue culture polystyrene dishes (TCPS) coated with collagen type I (Col), gelatin (Gel), or fibronectin (FN). The results revealed that the effective niches in each step were very different. The cardiac marker gene (GATA4, Tbx5, MEF2C) expression of iPSs at the 1st step was very high on the TCPS coated with FN or Gel, whereas on the FN-coated Es (especially with the 9 kPa modulus), the undifferentiated marker gene (Nanog) expression of iPSs was maintained. The expression of the contractile genes α-MHC, TnC1, and TnT2 and the self-beating (the 2nd step) of the NCMs were high on FN-coated TCPS and Col-coated Es. The 3rd step (beating duration) of the NCMs was effective on the Es, and at 21 days both the iPSs and NCMs stopped beating on the TCPS but were still beating on the Es. Overall, cardiac differentiation 'preferred' ECM-rigid culture substrates, and beating-behavior 'preferred' Col-soft culture substrates. These results are important for understanding and designing cardiac differentiation niches for regenerative medicine, and they suggest that a single culture substrate is not suitable for preparing self-beating cardiomyocytes.
The transplantation of beating cardiomyocytes (BCMs) is expected to be made more effective for serious heart diseases. The identification of the appropriate engineering processes and suitable culture substrates for inducing stem cell differentiation into BCMs is thus indispensable. The differentiation can be divided into three major processes, the cardiac differentiation step, the beating-induction step and the beating-duration step. A protocol with the higher efficiency in all of the steps must be useful. In this study, we separately evaluated the effect of culture substrates at each three step. We clarified that the biological and the physical properties of the culture substrates required at these steps were different. We found useful criteria for effective cardiac cell niche systems design.
干细胞为基础的心肌再生疗法已成为严重心脏病患者心脏移植的替代策略，但自体搏动成熟心肌细胞不可用。在这里，我们通过分别评估以下连续三个步骤来研究培养底物对诱导多能干细胞（iPS）体外心肌细胞分化的影响：（1）心肌标志物基因表达，（2）收缩基因表达和自我搏动，以及（3）搏动持续时间。为此，我们使用 iPS 细胞研究心脏分化，并使用新生大鼠心肌细胞（NCM）研究搏动行为。这些细胞在具有不同性质的底物上培养，即模量为 9、20 或 180 kPa 的弹性底物（Es），以及涂有 I 型胶原（Col）、明胶（Gel）或纤维连接蛋白（FN）的硬质组织培养聚苯乙烯盘（TCPS）。结果表明，每个步骤的有效生态位非常不同。第 1 步时 iPS 细胞的心肌标志物基因（GATA4、Tbx5、MEF2C）表达在涂有 FN 或 Gel 的 TCPS 上非常高，而在涂有 FN 的 Es 上（尤其是模量为 9 kPa 的 Es），iPS 细胞的未分化标志物基因（Nanog）表达得以维持。NCM 收缩基因 α-MHC、TnC1 和 TnT2 的表达和自我搏动（第 2 步）在 FN 涂覆的 TCPS 和 Col 涂覆的 Es 上较高。NCM 的第 3 步（搏动持续时间）在 Es 上有效，在第 21 天，iPS 和 NCM 都在 TCPS 上停止搏动，但仍在 Es 上搏动。总体而言，心肌分化“偏爱”细胞外基质刚性培养底物，而搏动行为“偏爱”Col 软性培养底物。这些结果对于理解和设计再生医学中的心脏分化生态位非常重要，并且表明单一培养底物不适合制备自发搏动的心肌细胞。
