title: Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system.
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.
authors.affiliation: Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, ROC.
authors.forename: Huai-En
authors.initials: HE
authors.lastname: Lu
authors.forename: Ming-Song
authors.initials: MS
authors.lastname: Tsai
authors.forename: Yao-Chen
authors.initials: YC
authors.lastname: Yang
authors.forename: Chen-Ching
authors.initials: CC
authors.lastname: Yuan
authors.forename: Tzu-Hao
authors.initials: TH
authors.lastname: Wang
authors.forename: Xi-Zhang
authors.initials: XZ
authors.lastname: Lin
authors.forename: Ching-Ping
authors.initials: CP
authors.lastname: Tseng
authors.forename: Shiaw-Min
authors.initials: SM
authors.lastname: Hwang
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism*; D000653:Amniotic Fluid / Q000166:cytology*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000201:enzymology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051379:Mice; D017154:Stromal Cells / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism
chemical_list: C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D000469:Alkaline Phosphatase