Viswanathan Sowmya, Benatar Tania, Mileikovsky Maria, Lauffenburger Douglas A, Nagy Andras, Zandstra Peter W
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 4 Taddle Creek Road, Toronto, Ontario M5S 3G9, Canada.
Biotechnol Bioeng. 2003 Dec 5;84(5):505-17. doi: 10.1002/bit.10799.
Although it is known that leukemia inhibitory factor (LIF) supports the derivation and expansion of murine embryonic stem (ES) cells, it is unclear whether this is due to inhibitory effects of LIF on ES cell differentiation or stimulatory effects on ES cell survival and proliferation. Using an ES cell line transgenic for green fluorescent protein (GFP) expression under control of the Oct4 promoter, we were able to simultaneously track the responses of live Oct4-GFP-positive (ES) and -negative (differentiated) fractions to LIF, serum, and other growth factors. Our findings show that, in addition to inhibiting differentiation of undifferentiated cells, the administration of LIF resulted in a distinct dose-dependent survival and proliferation advantage, thus enabling the long-term propagation of undifferentiated cells. Competitive responses from the differentiated cell fraction could only be elicited upon addition of serum, fibroblast growth factor-4 (FGF-4), or insulin-like growth factor-1 (IGF-1). The growth factors did not induce additional differentiation of ES cells, but rather they significantly improved the proliferation of already differentiated cells. Our analyses show that, by adjusting culture conditions, including the type and amount of growth factors or cytokines present, the frequency of media exchange, and the presence or absence of serum, we could selectively and specifically alter the survival, proliferation, and differentiation dynamics of the two subpopulations, and thus effectively control population outputs. Our findings therefore have important applications in engineering stem cell culture systems to predictably generate desired stem cells or their derivatives for various regenerative therapies.
虽然已知白血病抑制因子（LIF）可支持小鼠胚胎干细胞（ES细胞）的衍生和扩增，但尚不清楚这是由于LIF对ES细胞分化的抑制作用，还是对ES细胞存活和增殖的刺激作用。利用一种在Oct4启动子控制下表达绿色荧光蛋白（GFP）的ES细胞系，我们能够同时追踪活的Oct4-GFP阳性（ES）和阴性（分化）部分对LIF、血清及其他生长因子的反应。我们的研究结果表明，除了抑制未分化细胞的分化外，给予LIF还产生了明显的剂量依赖性存活和增殖优势，从而使未分化细胞能够长期传代培养。只有在添加血清、成纤维细胞生长因子-4（FGF-4）或胰岛素样生长因子-1（IGF-1）后，才能引发分化细胞部分的竞争性反应。这些生长因子并未诱导ES细胞进一步分化，而是显著提高了已分化细胞的增殖能力。我们的分析表明，通过调整培养条件，包括生长因子或细胞因子的类型和数量、培养基更换频率以及血清的有无，我们可以选择性且特异性地改变这两个亚群的存活、增殖和分化动态，从而有效控制群体产出。因此，我们的研究结果在构建干细胞培养系统以可预测地生成用于各种再生疗法的所需干细胞或其衍生物方面具有重要应用。
