Ward Christopher M, Eastham Angela M, Stern Peter L
Cancer Research UK Immunology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK.
Exp Cell Res. 2006 Jun 10;312(10):1713-26. doi: 10.1016/j.yexcr.2006.02.006. Epub 2006 Apr 17.
The 5T4 oncofoetal antigen is a cell surface glycoprotein that is transiently expressed during mouse ES cell differentiation and correlates with decreased pluripotency of such cells. We show that 5T4 antigen is transiently unregulated during HES4 and H1 human ES cell differentiation and its expression correlates with loss of the pluripotent markers OCT-4 and Tra-1-60 and upregulation of transcript markers associated with the three primary germ layers. To confirm that absence of cell surface 5T4 antigen represents a pluripotent hES cell phenotype, we performed mechanical transfer of either 5T4-ve or 5T4+ve HES4 colonies identified using live cell staining. 5T4-ve transfers maintained expression of OCT-4 in over 90% of resultant colonies, whereas 5T4+ve transfers exhibited significantly lower numbers of OCT-4-expressing colonies (92 +/- 1.4 vs. 2.9 +/- 2.0%). Interestingly, low cell density 5T4-ve colony transfers exhibited increased numbers of OCT-4-expressing colonies compared to large 5T4-ve transfers (92 +/- 1.4 vs. 63.2 +/- 1.9%). 5T4-ve and 5T4+ve HES4 and H1 ES cell lines expressed markers representative of neuroectoderm lineages, and we assessed the formation of neural lineages from these phenotypes in serum-containing medium and N2B27 medium. Expression of 5T4 was found to be inversely related to the yield of tyrosine-hydroxylase (TH+)-expressing neurons in N2B27 medium, with additional mesoderm and endoderm transcript markers detected. Homogeneous glial cell populations were derived from low cell density 5T4-ve colony transfers cultured in serum-containing medium, with TH+ neuronal formation inhibited in a cell-density-dependent manner. We conclude that the 5T4 antigen is a transient marker of hES cell differentiation and that 5T4 phenotype, colony seeding density and culture conditions significantly influence the maintenance of pluripotent hES cells and their differentiation to neural lineages.
5T4癌胚抗原是一种细胞表面糖蛋白，在小鼠胚胎干细胞分化过程中短暂表达，且与这些细胞多能性的降低相关。我们发现，在HES4和H1人胚胎干细胞分化过程中，5T4抗原短暂上调，其表达与多能性标志物OCT-4和Tra-1-60的丢失以及与三个原始胚层相关的转录标志物的上调相关。为了证实细胞表面5T4抗原的缺失代表多能性人胚胎干细胞表型，我们对通过活细胞染色鉴定的5T4阴性或5T4阳性HES4集落进行了机械转移。5T4阴性转移在超过90%的所得集落中维持OCT-4的表达，而5T4阳性转移中表达OCT-4的集落数量显著更低（92±1.4%对2.9±2.0%）。有趣的是，与大型5T4阴性转移相比，低细胞密度5T4阴性集落转移中表达OCT-4的集落数量增加（92±1.4%对63.2±1.9%）。5T4阴性和5T4阳性HES4和H1胚胎干细胞系表达代表神经外胚层谱系的标志物，我们在含血清培养基和N2B27培养基中评估了从这些表型形成神经谱系的情况。发现在N2B27培养基中，5T4的表达与表达酪氨酸羟化酶（TH+）的神经元产量呈负相关，同时检测到额外的中胚层和内胚层转录标志物。在含血清培养基中培养的低细胞密度5T4阴性集落转移产生了均匀的神经胶质细胞群体，TH+神经元的形成以细胞密度依赖的方式受到抑制。我们得出结论，5T4抗原是人胚胎干细胞分化的一个短暂标志物，并且5T4表型、集落接种密度和培养条件显著影响多能性人胚胎干细胞的维持及其向神经谱系的分化。
