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. 2018 May;7(2):87–93. doi: 10.5582/irdr.2018.01032

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2018, International Research and Cooperation Association for Bio & Socio - Sciences Advancement

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Figure 2. — iPSCs from a patient with phenylketonuria are pluripotent. (A) Test for absence of reprogramming vectors. RT-PCR of PKU-UiPSCs after 2 passages in (4) the positive control, urine cells (UCs) in (2) the negative control, (3) PKU-UiPSCs after 12 passages, H₂O in (1). (B) Alkaline phosphatase-positive ESC-like colonies (blue) from the PKU UCs (white). (C) Pluripotent UiPSCs (PKUC3-P10) and ESCs (HN4P60) expressed higher levels of the pluripotency markers OCT4, NANOG, and SOX2 in comparison to UCs (p < 0.05). (D) Flow cytometry of the pluripotency markers anti-stage specific embryonic antigen 4 (SSEA-4) (cell surface), anti-Tra-1-60 (cell surface), anti-Oct3/4 (nuclear), and anti- Tra-1-81 (cell surface). (E) Bisulfite genomic sequencing of the key pluripotent gene NANOG and OCT4 promoter area indicated a marked decrease in methylation in UiPSCs. The open and closed circles represent unmethylated and methylated CpGs, respectively. (F) Differentiated cultures of 7-day-old EBs (PKUC3-EB) expressed higher levels of 3 germ layer markers and lower levels of pluripotency markers in comparison to UiPSCs (PKUC3-P10) (p < 0.05).