Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, ROC.
Exp Cell Res. 2011 Aug 1;317(13):1895-903. doi: 10.1016/j.yexcr.2011.05.017. Epub 2011 May 26.
Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.
诱导多能干细胞（iPS）是通过外源表达四个转录因子，即 Oct4、Sox2、Klf4 和 c-Myc，也被称为山中因子，从体细胞中成功产生的。在实践中，最初的 iPS 集落是根据其胚胎干细胞（ES）细胞样形态选择的，但往往可能会在随后的检测中失败，例如碱性磷酸酶（AP）检测。在这项研究中，我们通过慢病毒递送共同表达了山中因子在羊水衍生（AF）细胞中。将 ES 样集落挑在传统饲养层上，发现部分或没有 AP 活性的高比例 AF-iPS。有趣的是，当集落首先接种在无饲养层培养系统上时，我们获得了绝大多数完全染色的 AP 阳性（AP+）AF-iPS 集落，然后转移到饲养层进行扩增。此外，未检测到无 AP 活性的集落。这一筛选步骤减少了形态学和 AP 检测之间的差异。我们观察到在饲养层上生长的 AF-iPS 集落中有 28%的 AP+集落，45%的 AP 部分阳性（AP+/-）集落和 27%的 AP 阴性（AP-）集落，而在无饲养层系统筛选的集落中有 84%的 AP+集落，16%的 AP+/-集落和没有 AP-集落。通过无饲养层筛选的 AP+AF-iPS 集落也表达多能标记物 OCT4、SOX2、NANOG、TRA-1-60、TRA-1-81、SSEA-3 和 SSEA-4，并且具有体外和体内向三个胚层分化的能力。在这项研究中，我们报告了一种简单的、一步法通过无饲养层筛选选择 AP+AF-iPS 细胞的方法。
