Dhara Sujoy K, Hasneen Kowser, Machacek David W, Boyd Nolan L, Rao Raj R, Stice Steven L
Regenerative Bioscience Center, University of Georgia, Athens, GA 30605, USA.
Differentiation. 2008 May;76(5):454-64. doi: 10.1111/j.1432-0436.2007.00256.x. Epub 2008 Jan 3.
Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.
已有报道称，在培养过程中可利用饲养层细胞或条件培养基从人胚胎干细胞（hES）中衍生出人神经祖细胞（hNP）。这会将未定义成分引入系统，限制了精确研究控制该过程的因子需求的能力。此外，使用非人类来源的饲养层细胞存在人畜共患病传播的可能性，限制了其临床应用价值。在此，我们报告一种无饲养层系统，用于从hES细胞中产生hNP，并测试该过程中各种培养基成分的作用。比较了使用定义培养基成分的五种方案在hNP生成效率方面的差异。基于此分析，我们讨论了碱性成纤维细胞生长因子（FGF2）、N2添加剂、非必需氨基酸（NEAA）和 KnockOut血清替代物（KSR）在hNP生成过程中的作用。所有方案均导致Oct4/POU5F1表达下调（从90.5%降至<3%），并不同程度地上调神经祖细胞标志物。含有N2但不含KSR和NEAA的培养基所产生的培养物中，神经祖细胞标志物Musashi 1（MSI1）的表达显著更高（p<0.05）。3周后，这些细胞中约89%为巢蛋白（NES）阳性，但它们不增殖。相比之下，添加KSR和NEAA的分化培养基产生的NES阳性细胞较少（75%），但这些细胞具有增殖能力，到第五代时培养物中>97%为NES阳性细胞。这表明KSR和NEAA添加剂并未增强早期分化，但确实促进了hNP细胞培养物的增殖。这产生了一种高效、稳健、可重复的分化系统，适用于生成大量hNP细胞。这将有助于进一步研究人类早期神经分化中的分子和生化机制，并有可能产生用于治疗用途的均匀神经元细胞，而无需担心来自饲养层的人畜共患病传播。
