Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures
Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures
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DOI：
10.1634/stemcells.2004-0184
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发表时间：
2005-06-01
期刊：
影响因子：
5.2
通讯作者：
Mummery, C
中科院分区：
文献类型：
--
作者：
Passier, R;Oostwaard, DWV;Mummery, C
人胚胎干细胞（hESCs）可以分化为心肌细胞，但分化效率较低。我们在20%胎牛血清（FCS）存在的情况下，通过与内脏内胚层样细胞系END-2共培养，常规诱导HES-2细胞系心肌细胞分化。在这项研究中，我们证明了在HES-2-END-2共培养过程中心肌细胞分化与FCS浓度之间存在显著的反比关系。与添加20% FCS相比，不添加FCS时共培养中跳动区域的数量增加了24倍。当在无血清共培养中添加抗坏血酸时，观察到跳动区域的数量增加了40%。无血清共培养量的增加伴随着心脏标志物和心脏祖细胞标志物Isl1 mRNA和蛋白表达的增加。在HES-2与END-2细胞共培养后12天，跳动区数量增加。然而，在无血清共培养（503 +/- 179；平均标准误差）和20% FCS共培养（312 +/- 227）之间，每个跳动区域α -肌动素阳性心肌细胞的数量没有显著差异。无血清共培养对心肌细胞分化的刺激作用不仅见于HES-2，而且见于HES-3和HES-4细胞系。为了在未来产生足够的心肌细胞用于细胞替代治疗，从hESCs中扩大心肌细胞的形成是必不可少的。目前的数据向这个方向迈出了一步，并代表了一种改进的体外模型，没有血清中的干扰因素，用于测试可能促进心肌细胞分化的其他因素。
Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is low. We routinely induce cardiomyocyte differentiation of the HES-2 cell line by coculture with a visceral endoderm-like cell line, END-2, in the presence of 20% fetal calf serum (FCS). In this study, we demonstrate a striking inverse relationship between cardiomyocyte differentiation and the concentration of FCS during HES-2-END-2 coculture. The number of beating areas in the cocultures was increased 24-fold in the absence of FCS compared with the presence of 20% FCS. An additional 40% increase in the number of beating areas was observed when ascorbic acid was added to serum-free cocultures. The increase in serum-free cocultures was accompanied by increased mRNA and protein expression of cardiac markers and of Isl1, a marker of cardiac progenitor cells. The number of beating areas increased up to 12 days after initiation of coculture of HES-2 with END-2 cells. However, the number of alpha-actinin-positive cardiomyocytes per beating area did not differ significantly between serum-free cocultures (503 +/- 179; mean standard error of the mean) and 20% FCS cocultures (312 +/- 227). The stimulating effect of serum-free coculture on cardiomyocyte differentiation was observed not only in HES-2 but also in the HES-3 and HES-4 cell lines. To produce sufficient cardiomyocytes for cell replacement therapy in the future, upscaling cardiomyocyte formation from hESCs is essential. The present data provide a step in this direction and represent an improved in vitro model, without interfering factors in serum, for testing other factors that might promote cardiomyocyte differentiation.