Cytotechnology. 2004 Jan;44(1-2):77-91. doi: 10.1023/B:CYTO.0000043414.90681.c2.
Embryonic stem (ES) cells have been in the fore front of scientific literature lately as having the potential for regeneration of many tissue types. Two important issues that need to be addressed are the culture conditions for maintaining ES cells and the accuracy of ES cell markers in monitoring the undifferentiated state. Leukaemia inhibitory factor (LIF) is routinely used to sustain mouse ES cells (mES) in a pluripotent fashion. In this paper, we assessed three markers during long-term maintenance of ES cells with various concentrations of LIF to see if decreasing concentration would lead to changes in marker expressions and growth behavior. Common markers of pluripotency such as alkaline phosphatase enzyme activity (ALP), surface staining for stage specific embryonic antigen 1 (SSEA-1), Oct-4 transcription factor, cell doubling time, as well as visual observations of cell morphology were analyzed during long-term maintenance of mES cells with LIF concentrations ranging from 0 to 500 pM. The morphology of the cells at LIF concentrations of 0 25 pM changed from being tight clusters to more flattened shapes while cells in 50-500 pM retained the clustered shape but growth rates remained essentially identical at between 10 and 16 h. ES cells at all concentrations of LIF continued expressing ALP, SSEA-1 and Oct-4 markers over a period of 6 weeks, which indicate that mES cells are capable of either producing autocrine LIF or are able to proliferate at very low levels of LIF. Pluripotency markers such as Oct-4 and SSEA-1 are only moderately reduced after 5-6 weeks. Oct-4 mRNA expression levels were partially diminished in LIF free conditions only at weeks 5 and 6 compared to controls with LIF at 500 pM. Changes in morphology of cells by visual observation seemed to be a faster indication of the onset of differentiation in mES cells, although other reliable means also include decreased levels of Oct-4, SSEA-1 and ALP markers. It is preferable to maintain long-term cultures of mES cells above 50 pM of LIF to have a more homogenous, stable population of pluripotent cells.
胚胎干细胞（ES 细胞）最近成为科学文献的前沿，因为它们具有再生多种组织类型的潜力。需要解决的两个重要问题是维持 ES 细胞的培养条件和 ES 细胞标志物在监测未分化状态中的准确性。白血病抑制因子（LIF）通常用于维持多能状态的小鼠 ES 细胞（mES 细胞）。在本文中，我们评估了 LIF 浓度不同时 ES 细胞长期维持的三种标志物，以观察浓度降低是否会导致标志物表达和生长行为的变化。碱性磷酸酶酶活性（ALP）、阶段特异性胚胎抗原 1（SSEA-1）表面染色、Oct-4 转录因子、细胞倍增时间等多能性常见标志物，以及细胞形态的直观观察，均在 LIF 浓度范围为 0 至 500 pM 的 mES 细胞长期维持过程中进行了分析。在 0 至 25 pM 的 LIF 浓度下，细胞形态由紧密聚集变为更扁平的形状，而在 50 至 500 pM 的细胞保持聚集形状，但生长速度基本相同，在 10 至 16 小时之间。所有 LIF 浓度的 ES 细胞在 6 周内继续表达 ALP、SSEA-1 和 Oct-4 标志物，这表明 mES 细胞能够产生自分泌 LIF 或能够在非常低的 LIF 水平下增殖。多能性标志物如 Oct-4 和 SSEA-1 在 5-6 周后仅适度减少。与 500 pM LIF 对照相比，仅在第 5 和第 6 周，无 LIF 条件下 Oct-4mRNA 表达水平部分降低。细胞形态的变化通过肉眼观察似乎是 mES 细胞分化开始的更快指示，尽管其他可靠手段还包括 Oct-4、SSEA-1 和 ALP 标志物水平的降低。最好将 mES 细胞的长期培养维持在 50 pM 以上的 LIF 水平，以获得更同质、稳定的多能细胞群体。
