Du X X, Shi W K
Shanghai Institute of Cell Biology, Academia Sinica, China.
Shi Yan Sheng Wu Xue Bao. 1996 Dec;29(4):413-27.
We constructed plasmids pSVLD(+) and pSVLD(-) containing human D-form Leukemia Inhibitory Factor (LIF) cDNA sequence in sense or antisense orientation, transfected them into cells of an embryonic stem cell line ES-5, and isolated 248 pSVLD(+)-transfected and 93 pSVLD(-)-transfected G 418-resistant clones. By stepwise reducing LIF concentration in the medium, we obtained 3 pSVLD(+)-transfected clones (A 1-3) that could grow in 15% BRL-CM, including ESL(+)A 2 that could grow without LIF: we also obtained 13 pSVLD(-)-transfected clones (B 1-13) which would differentiate in 60% BRL-CM, including ESL(-)B 3 and B 5 that could not be passaged without LIF. ESL(+)A 2 and ESL(-)B 5 cells had the relatively stronger LIF mRNA or antisense LIF RNA expression, and LIF overexpression in ESL(+)A 2 cells was shown by biological assay for ES cell differentiation inhibition. ESL(+)A 2 cells could be continuously passaged for at least 13 passages without addition of exogenous LIF, retained undifferentiated morphology as well as a high growth rate, and resembled ES-5 cells in terms of stem cell characteristics and pluripotent properties, as analyzed for alkaline phosphatase activity and with staining the paraffin sections of tumor formed by inoculating ESL(+) A 2 cells into mouse. On the contrary, ESL(-) cells should be cultured in higher concentration of LIF than ES-5 cells, otherwise, would undertake extensive differentiation. By hanging drop culture for 3 days in the presence of 10(-6) mol/L RA then observing the differentiation of the formed embryonic bodies (EBs), we found that ESL(+) A 2 and ES-5 cells underwent similar morphologically differentiation, with round and epitheliallike cells occurring around the EBs; while ESL(-) B 5 cells, despite initial differentiation to round cells, differentiate into fibroblast-like and spindle shaped cells. The above results indicate that LIF overexpression in ESL(+) A 2 cells is able to completely free ES cells from the dependence on LIF-conditioned medium, and endogenous LIF gene expression, although is very low, may be indispensable for inhibiting the differentiation in vitro of ES cells; LIF overexpression might not obviously change the differentiation way of ES-5 cells, however, blocking endogenous LIF expression gives rise to the increased sensitivity of ES-5 cells to differentiate, with an altered differentiation pattern. The establishment of ESL(+) and ESL (-) cell lines provides models for further study of the growth and differentiation of ES-5 cells.
我们构建了质粒pSVLD(+)和pSVLD(-)，它们含有处于正义或反义方向的人D型白血病抑制因子（LIF）cDNA序列，将其转染到胚胎干细胞系ES-5的细胞中，并分离出248个转染pSVLD(+)的和93个转染pSVLD(-)的G418抗性克隆。通过逐步降低培养基中LIF的浓度，我们获得了3个转染pSVLD(+)的克隆（A1-3），它们可以在15%的BRL-CM中生长，其中包括ESL(+)A2，它可以在无LIF的情况下生长；我们还获得了13个转染pSVLD(-)的克隆（B1-13），它们在60%的BRL-CM中会发生分化，其中包括ESL(-)B3和B5，它们在无LIF的情况下无法传代。ESL(+)A2和ESL(-)B5细胞具有相对较强的LIF mRNA或反义LIF RNA表达，并且通过ES细胞分化抑制的生物学测定表明ESL(+)A2细胞中LIF过表达。ESL(+)A2细胞可以在不添加外源性LIF的情况下连续传代至少13代，保持未分化的形态以及高生长速率，并且在碱性磷酸酶活性分析以及对将ESL(+)A2细胞接种到小鼠体内形成的肿瘤石蜡切片染色方面，在干细胞特征和多能性方面类似于ES-5细胞。相反，ESL(-)细胞应该在比ES-5细胞更高浓度LIF的条件下培养，否则会发生广泛分化。通过在10(-6)mol/L RA存在下悬滴培养3天，然后观察形成的胚体（EBs）的分化，我们发现ESL(+)A2和ES-5细胞经历了相似的形态学分化，EBs周围出现圆形和上皮样细胞；而ESL(-)B5细胞，尽管最初分化为圆形细胞，但会分化为成纤维细胞样和纺锤形细胞。上述结果表明，ESL(+)A2细胞中LIF过表达能够使ES细胞完全摆脱对LIF条件培养基的依赖，并且内源性LIF基因表达，尽管非常低，但可能对于抑制ES细胞体外分化是不可或缺的；LIF过表达可能不会明显改变ES-5细胞的分化方式，然而，阻断内源性LIF表达会导致ES-5细胞对分化的敏感性增加，分化模式改变。ESL(+)和ESL(-)细胞系的建立为进一步研究ES-5细胞的生长和分化提供了模型。
