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.