CEINGE-Biotecnologie Avanzate, Naples, Italy.
Cytotherapy. 2011 Mar;13(3):350-6. doi: 10.3109/14653249.2010.520311. Epub 2010 Sep 27.
Procedures for cardiomyocyte differentiation of mouse embryonic stem cells (mESCs) utilize different amounts of serum. Because the serum composition is unknown, unambiguous characterization of the differentiation process is biased. All reported serum-free protocols used for compound testing provide serum throughout the differentiation process. We report on an embryoid body (EB)-based procedure for cardiomyocyte differentiation of mESCs in which serum is provided only in the earliest step (hanging drop, 0-2 days).
To assess cardiomyocyte differentiation, we generated an mESCs clone that expressed green fluorescence protein (GFP) under the control of the myosin light chain 2v (MLC2v) promoter. To define the lowest serum concentration required for efficient induction of cardiomyocyte differentiation, EBs were formed in presence of 5% (S5), 10% (S10) and 15% (S15) serum until day 2, then switched to a serum-free medium.
Analysis of cardiac-specific transcripts on day 6 of differentiation showed that 10% (S10) was the minimum amount of serum for efficient continuation of cultures under serum-free conditions. Spontaneously beating foci were detected in 90.0 ± 5.5% of S10 EBs on day 7 of differentiation, and cardiomyocyte markers were expressed from day 8 of differentiation (MLC2v-driven GFP; α-myosin heavy chain). Dose-response curves to isoproterenol showed that the beating rate increased by 113.0 ± 39.4%, with a concentration for half-maximal effect (EC(50)) of 25.7 nm.
The development of functional cardiomyocytes from mESCs is not affected by serum withdrawal after EBs formation. This culture system represents a new model for cardiomyocyte differentiation of mESCs to assess the effects of compounds on the process of cardiomyogenesis.
小鼠胚胎干细胞（mESCs）的心肌细胞分化程序需要使用不同量的血清。由于血清成分未知，因此对分化过程的明确描述存在偏差。所有已报道的无血清协议都在分化过程中提供血清。我们报告了一种基于胚状体（EB）的 mESC 心肌细胞分化程序，其中仅在最初的步骤（悬滴，0-2 天）中提供血清。
为了评估心肌细胞分化，我们生成了一个 mESC 克隆，该克隆在肌球蛋白轻链 2v（MLC2v）启动子的控制下表达绿色荧光蛋白（GFP）。为了确定诱导心肌细胞分化所需的最低血清浓度，我们在 5%（S5）、10%（S10）和 15%（S15）血清中形成 EB，直到第 2 天，然后切换到无血清培养基。
分化第 6 天分析心脏特异性转录本显示，10%（S10）是在无血清条件下高效维持培养的最低血清量。分化第 7 天检测到 90.0±5.5%的 S10 EB 中自发搏动焦点，从分化第 8 天开始表达心肌细胞标志物（MLC2v 驱动的 GFP；α-肌球蛋白重链）。异丙肾上腺素的剂量反应曲线显示，搏动率增加了 113.0±39.4%，半最大效应浓度（EC50）为 25.7nm。
EB 形成后血清撤离不会影响 mESC 向功能性心肌细胞的发育。这种培养系统代表了评估化合物对心肌发生过程影响的 mESC 心肌细胞分化的新模型。
