Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.
Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.
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DOI：
10.1371/journal.pone.0086031
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Zhao Y
中科院分区：
文献类型：
--
作者：
Quang T;Marquez M;Blanco G;Zhao Y
为了扩增人类多能干细胞(hESCs和hiPSCs)，已经开发了许多不同的培养系统。一般来说，在依赖饲养层的系统中，无论饲养层细胞类型如何，都需要在培养基中添加4-10 ng/ml的碱性成纤维细胞生长因子，而在无饲养层的系统中，为了在各种底物上保持长期培养，需要高达100 ng/ml的碱性成纤维细胞生长因子。在天然的hESCs生长环境中所需的bFGF的数量尚不清楚。在此，我们用灭活的人脂肪间充质干细胞作为饲养细胞，观察了两个hESCs系(H1和H9)和一个hPSCs系(DF19-9-7T)在添加0、0.4或4 ng/mlbFGF的培养液中长期平行培养长达23代的情况，以及H9和DF19在添加4、20或100 ng/mlbFGF的培养液中长达13代的平行培养作为比较。在所测试的所有细胞系中，bFGF补充剂以剂量依赖的方式显示出对生长扩张、单细胞定植和冻存恢复的抑制作用。此外，碱性成纤维细胞生长因子对不同的细胞株有不同的作用，在4 ng/ml或更高的浓度下诱导H1和DF19分化，而允许H9在相同浓度下长期培养，没有明显的剂量效应。除H1-4 ng外，0、0.4和4 ng/mlbFGF组及4、20和100 ng/mlbFGF4、20和100 ng/mlbFGF组细胞系均具有多能性。然而，DF19在0和4 ng/mlbFGF液中显示相似的核型异常，而在长期培养的0 ng/mlbFGF液中H1和H9核型正常。结果表明，外源性bFGF对间充质干细胞上培养的人多能干细胞具有剂量和细胞系依赖性的作用，提示在hESCs/hiPSCs培养中bFGF的最佳使用应根据特定的细胞系和培养体系而定。
Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.
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影响因子：
14.8
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