Title: Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system
Authors: Lu, Huai-En
Tsai, Ming-Song
Yang, Yao-Chen
Yuan, Chen-Ching
Wang, Tzu-Hao
Lin, Xi-Zhang
Tseng, Ching-Ping
Hwang, Shiaw-Min
Department of Biological Science and Technology
Keywords: Amniotic fluid;Induced pluripotent stem cells;Feeder free;Alkaline phosphatase
Issue Date: 1-Aug-2011
Abstract: 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. (C) 2011 Elsevier Inc. All rights reserved.
URI: http://dx.doi.org/10.1016/j.yexcr.2011.05.017
ISSN: 0014-4827
DOI: 10.1016/j.yexcr.2011.05.017
Volume: 317
Issue: 13
Begin Page: 1895
End Page: 1903
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