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T. Yoshida, H. Koizumi, K. Yuki, K. Tsubota, Y. Ozawa, H. Okano; Purification of Human iPS Cells From Feeder Cells to Analyze DNA Sequence. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2608.
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© ARVO (1962-2015); The Authors (2016-present)
To establish the method for analyzing whole DNA sequence of human iPS cells. This study will be the first step for the regenerative medicine, since the genomic information will be important in applying the iPS cells for transplantation.
Human iPS cells were cultured on the feeder cells, STO cells, derived from mouse embryonic fibroblast (MEF). Whole cells were dissociated into single cells by incubating with 0.05% trypsin EDTA to perform flow cytometry. To purify the human iPS cells, following 2 methods were compared; (1) The total cells were sorted by forward scatter (FSC) and side scatter (SSC), to distinguish the cells by their size. (2) The total cells were incubated with Tra-1-60 antibody, which recognizes human tetracarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES), and sorted. Purification rate was evaluated by real time PCR using two pairs of primer sets; One specifically recognizes human genome, and another, mouse genome.
(1) The FACS patterns of both iPS cells including STO cells, and the STO cells only, were almost the same. Thus, the cells were not separated by this method. (2) Tra-1-60 positive cells were 73% of the living cells. Real time PCR showed that the ratio of human cells in this fraction was 99.5%.
Flow cytometry using Tra-1-60 antibody was useful for the purification of human iPS cells from STO cells. This method will allow us the future analysis of whole DNA sequence of iPS cells with the least reading errors.
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