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. 2020 Aug 17;11:4110. doi: 10.1038/s41467-020-17901-2

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© The Author(s) 2020

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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Fig. 8 — a Cells were cultured under flow conditions for 4 days and the expression of heparan sulfate was evaluated by immunofluorescence. Intensity of heparan sulfate was calculated in each picture and normalized by cell number. The normalized fluorescence intensity at day 4 was divided with the one at day 0. Scale bar is 10 μm. n > 4 images examined over six independent experiments. Statistical analyses were performed by one-way ANOVA followed by Newman–Keuls’s post test. b Gene expression of glycocalyx markers (SDC1: syndecan 1, SDC2: syndecan 2, SDC4: syndecan 4, GPC: glypican, PLC: perlecan), as evaluated by qRT-PCR, in HGPS-iPSC SMCs cultured under flow conditions. Gene expression was normalized by the housekeeping gene GAPDH, and the normalized gene expression at day 4 divided by day 0. n = 3 technical replicates from a pool of three independent experiments. c HGPS-iPSCs-SMC cultured under flow condition were treated or not with heparinase III and the number of cells per microfluidic area during culture was calculated and normalized by the number of cells present at day 2. n = 3 independent experiments. Statistical analyses were performed by a two-tailed unpaired Student’s t test. d Quantification of MMP13 activity (cell culture media) by ELISA. Cells were analyzed at day 4 under flow. Fluorescence signal was normalized by cell number and then by control experimental group. n > 9 images examined over six independent experiments. Statistical analyses were performed by a two-tailed unpaired Student’s t test. e Expression of alkaline phosphatase in HGPS-iPSCs SMC, normalized by cell number per mm², in cells cultured 4 days under flow conditions. Cells were treated or not with heparinase III. n = 2 technical replicates over three independent experiments. Statistical analyses were performed by a two-tailed unpaired Student’s t test. In a–e, results are mean ± SEM. *, **, ***, **** denote statistical significance (p < 0.05, p < 0.01, p < 0.001, p < 0.0001). f Summary of the results.