Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system

Huai En Lu, Ming Song Tsai, Yao Chen Yang, Chen Ching Yuan, Tzu Hao Wang, Xi Zhang Lin, Ching-Ping Tseng, Shiaw Min Hwang*

*Corresponding author for this work

Research output: Contribution to journalArticle

21 Scopus citations

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.

Original languageEnglish
Pages (from-to)1895-1903
Number of pages9
JournalExperimental Cell Research
Volume317
Issue number13
DOIs
StatePublished - 1 Jan 2011

Keywords

  • Alkaline phosphatase
  • Amniotic fluid
  • Feeder free
  • Induced pluripotent stem cells

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