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. 2019 Feb 6;9:1490. doi: 10.1038/s41598-018-37291-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The changes of ALP activity in human skin-derived fibroblasts after repeated transfection. (a) Time-line for assessment of pluripotency-related gene expression in fibroblasts after repeated transfection. (b) Cytochemical evaluation of ALP activity in the fibroblasts after repeated transfection with the reprogramming factors. Fibroblasts were transfected with Yamanaka’s four reprogramming factors once or twice. The treated cells were subjected to cytochemical staining for ALP activity at 3, 5, 7, and 9 days after the final transfection, as shown in (a). Bar = 500 μm. (c) Ratio of ALP-positive cells and (d) proliferation curves for fibroblasts when the parental fibroblasts were subjected to transfection once or twice. After the final transfection, cells were seeded onto 4- or 24-well plate, and fixed (for cytochemical staining for ALP activity) or harvested (for counting the cell number) at the days indicated.