Basic fibroblast growth factor support of human embryonic stem cell self-renewal
Basic fibroblast growth factor support of human embryonic stem cell self-renewal
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DOI：
10.1634/stemcells.2005-0247
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发表时间：
2006-03-01
期刊：
影响因子：
5.2
通讯作者：
Thomson, James A.
中科院分区：
文献类型：
--
作者：
Levenstein, Mark E.;Ludwig, Tenneille E.;Thomson, James A.
人胚胎干 (ES) 细胞最常在碱性成纤维细胞生长因子 (FGF2) 存在的情况下在成纤维细胞饲养层或成纤维细胞条件培养基中培养。最近有报道称，升高浓度的 FGF2 允许在没有成纤维细胞或成纤维细胞条件培养基的情况下培养人 ES 细胞。在此，我们比较了补充有 4、24、40、80、100 和 250 ng/ml FGF2 的无条件培养基 (UM) 通过多次传代维持低密度人 ES 细胞培养物的能力。在这些严格的培养条件下，4、24 和 40 ng/ml FGF2 无法维持人 ES 细胞传代 3 代，但 100 ng/ml 可以维持人 ES 细胞，其有效性与条件培养基 (CM) 相当。两种人 ES 细胞系（H1 和 H9）在补充有 100 ng/ml FGF2 的 UM 中维持多达 164 次群体倍增（7 个月和 4 个月）。经过长时间培养，这些细胞被注射到严重联合免疫缺陷米色小鼠体内时形成畸胎瘤，并表达未分化的人类 ES 细胞的特征标记。我们还证明 FGF2 在 UM 中比在 CM 中降解得更快，部分解释了 UM 中需要更高浓度的 FGF2。这些结果进一步促进了人类 ES 细胞的大规模常规培养，并表明成纤维细胞和成纤维细胞条件培养基部分通过将 FGF 信号稳定在临界阈值之上来维持人类 ES 细胞。
Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic fibroblast growth factor (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. It has recently been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Herein we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100, and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture, the cells formed teratomas when injected into severe combined immunodeficient beige mice and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells and suggest that fibroblasts and fibroblast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold.