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. 2024 Sep 10;25(10):4433–4464. doi: 10.1038/s44319-024-00243-1

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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Figure EV4 — (A) Left: Quantification of FUT1 transcripts in WT, mock-transfected (control ECs), and FUT1-transfected (FUT1⁺ECs), WA09 hESCs in 3 clones for each line after transfection. Right: Representative histograms showing relative counts and MFI of WT hESCs and FUT1⁺ECs expressing α-fucose. n = 3 technical replicates for each line. (B) Quantification of representative genes of the tri-germ layers, NKX2.5, SOX17, and PAX6, in WT hESCs and in 3 clones of control ECs and FUT1⁺ECs for each line showing unchanged genes after transfection. (C) Left and center: Representative brightfield and endogenous fluorescence protein (red, left) and (green, center) images showing clone of control ECs 18 d after culturing sorted cells. Right: Representative bright-field and endogenous fluorescence protein (green) image showing clone of FUT1⁺ECs 18 d after culturing sorted cells. Scale bars represent 100 μm. n = 3 technical replicates. (D) Heatmap of subset of RNA-seq-based gene expression profiles showing low expression of FUT1 1 d after H7 hESC differentiation into tissues that differentially expressed GT genes. Anterior primitive streak (APS), day 1 mid PS (MPS), and day 2 LM, PM, DLL^-, and DLL⁺, PM cells. n = 3 per group. (E) Quantification of the mRNA expression levels of FUT1 in WT hESCs, control ECs, and FUT1⁺ECs on day 0 and after differentiation into LM for 3 d showing high and constant expression of FUT1. (F) Quantification of the mRNA expression levels of FUT1 in WT hESCs and FUT1⁺ECs on day 0 and after differentiation into DE for 3 d showing high and constant expression of FUT1. n = 3 technical replicates. Error bars represent ± SDs. (G) Representative histograms of relative counts and MFI of WT hESCs and FUT1⁺ECs showing the expression of α-fucose residues in WT hESCs and FUT1⁺ECs after differentiation into NPCs for 3 d and 6 d. (H) Quantification of NE markers showing SOX1 and OTX2 mRNA expression in WT hESCs, control ECs and FUT1⁺ECs on day 0 and after differentiation into NPCs for 3 d. In all experiments, n = 3 technical replicates. Data information: In (A, G), data are presented as means ± SD. Ordinary One-way ANOVA **P < 0.01, ***P < 0.001. In (A, B, E, H), data are presented as means ± SEM. Two-tailed Student’s t-tests ****p < 0.0001 or non-significance (NS).