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. 2019;7(3):686-688.e5.
doi: 10.1016/j.jcmgh.2018.11.001. Epub 2018 Dec 10.

Human Norovirus Propagation in Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Cells

Shintaro Sato  1 , Kota Hisaie  2 , Shiho Kurokawa  3 , Akio Suzuki  4 , Naomi Sakon  5 , Yohei Uchida  3 , Yoshikazu Yuki  3 , Hiroshi Kiyono  6
Affiliations

Human Norovirus Propagation in Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Cells

Shintaro Sato et al. Cell Mol Gastroenterol Hepatol. 2019.
No abstract available

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Figures

Figure 1
Figure 1
Replication of HuNoVs in IECs derived from human iPSCs. Monolayered human iPSC–derived IECs were inoculated with 2 × 106 genome equivalents of the indicated HuNoV genotypes. Inoculation and sampling were done as described in the Materials and Methods. Viral genome RNA was extracted from both supernatants (ie, those taken at 3 and 72 hours postinfection [hpi]), and then genome equivalents were quantified with reverse transcriptase quantitative polymerase chain reaction. Samples at 3 hpi were used as references. Each value is representative of at least 3 independent experiments and is shown as the mean ± SD from between 4 and 6 wells of supernatants of each culture group.
Figure 2
Figure 2
Replication of GII.4 genotype HuNoVs is prevented by pretreatment with either anti-GII.4 or anti-GII.17 pAbs. Before inoculation of IECs, 2 × 106 genome equivalents of each HuNoV genotype was incubated with 100 ng of (A) anti-GII.4 or (B) anti-GII.17 Ab, or with normal rabbit immunoglobulin G, for 1.5 hours. Inoculation, sampling, and quantification of genome equivalents were done as described in the Materials and Methods. Each value is representative of at least 3 independent experiments and is shown as the mean ± SD from between 4 and 6 wells of supernatants of each culture group. *P < .05. hpi, hours postinfection.
Supplementary Figure 1
Supplementary Figure 1
Polarized monolayer of IECs derived from human iPSCs. (A) Differentiation of iPSC-derived IECs to enterocytes. Relative messenger RNA expression of the indicated genes in iPSC-derived IECs during the course of differentiation (days 0, 2, and 4) were determined by qPCR and normalized against the expression of GAPDH. The assays were performed in 3 independent biological replicates. Expression levels are presented as means ± SEM. SI, sucrase isomaltase. (B, D) Immunohistochemical analysis of monolayers harvested with the Transwell membrane after 6 days of culture. Sections were stained with anti-E-cadherin (green) and anti-villin-1 antibodies (red, B) or Ulex europaeus agglutinin-1 (red, D), and counterstained with DAPI (blue). Data are representative of 3 independent experiments. Scale bar = 10 μm. HBGA, histo-blood group antigen. (C) Transmission electron microscopic images of polarized monolayer of human iPSC–derived IECs. Lower panel is a magnification of the area in the blue box in the upper panel. Data are representative of 3 independent experiments. Scale bar = 2.5 μm. (E, F) GII.4 virus and VLPs bound to, and entered, the iPSC-derived IECs. Polarized iPSC-derived IECs on Transwell membranes were incubated with 8.3 × 108 genome equivalents of GII.4 virus or 300 ng of VLPs for 3 hours, and then the Transwell membranes were whole-mount stained with anti-GII.4 antibody (E, red) or were sectioned and stained with anti-GII.4 antibody (F, red) simultaneously with anti-villin-1 antibody (F, green), and then counterstained with DAPI (blue). Lower panels are a magnification of the area in the yellow boxes in the upper panels. Data are representative of three independent experiments. Scale bar = 50 μm (E), 10 μm (F).
Supplementary Figure 2
Supplementary Figure 2
Inactivation of HuNoVs by heat and sodium hypochlorite treatment. Monolayered human iPSC–derived IECs were inoculated with (A) 2 × 106 or (B) 3.7 × 107 (GII.4) or 5.8 × 108 (GII.17) genome equivalents of the indicated HuNoV genotypes. Inoculation and sampling were done as described in the Materials and Methods. Viral genome RNA was extracted from each supernatant sampled at (A) the indicated time or (B) 24 to 48 hpi, and then genome equivalents were quantified with reverse transcriptase qPCR. Each value is representative of at least 3 independent experiments and is shown as the mean ± SD from (A) 6 or (B) 3 wells of supernatants of each culture group. (C) GII.4 and GII.17 HuNoVs (2 × 106 genome equivalents) were incubated at 60°C for the indicated times. (D) GII.4 HuNoVs (2 × 107 genome equivalents) were incubated with the indicated concentrations of sodium hypochlorite (NaClO) at room temperature for 30 minutes, and then diluted with base medium to 2 × 106 genome equivalents/100 μL. Monolayered human iPSC–derived IECs were inoculated with each treated HuNoV. Viral genome RNA was extracted from both supernatants, and the genome equivalents were quantified by reverse transcriptase qPCR. Samples at 3 hpi were used as in the references (A, C, and D). Each value is representative of 3 independent experiments and is shown as the mean ± SD from 6 wells of supernatants of each culture group. *P < .05.
Supplementary Figure 3
Supplementary Figure 3
Binding of GII.4 and GII.17 VLPs to histo-blood group antigens is prevented by pre-treatment with each anti-VLP polyclonal antibody (pAb). (A) Homotypic titer of anti-GII.4 and GII.17 pAbs were quantified by enzyme-linked immnosorbent assay. Data are means ± SD from 1 experiment representative of 2 independent experiments. (B) Blocking activity of anti-GII.4 and anti-GII.17 toward VLP–histo-blood group antigen binding was measured by ELISA. Data are mean ± SD from 1 experiment representative of 3 independent experiments. *P < .05. GII.3 and GII.6 HuNoVs (2 × 106 genome equivalents) were incubated with 100 ng of (C) anti-GII.4 or (D) anti-GII.17 antibody, or with normal rabbit IgG, for 1.5 hours. Monolayered human iPSC–derived IECs were inoculated with each treated HuNoV. Viral genome RNA was extracted from both supernatants, and the genome equivalents were quantified by reverse transcriptase qPCR. Samples at 3 hpi were used as references. Each value is representative of 3 independent experiments and is shown as the mean ± SD from 4 to 6 wells of supernatants of each culture group.
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References

    1. Ettayebi K. Science. 2016;353:1387–1393. - PMC - PubMed
    1. Forbester J.L. Humana Press; 2016.
    1. Rankin S.A. Nature. 2011;470:105–109. - PMC - PubMed
    1. Takahashi Y. Stem cell reports. 2018;10:314–328. - PMC - PubMed
    1. Takahashi Y. EBioMedicine. 2017;23:34–45. - PMC - PubMed

Supplementary References

    1. Takayama N. J Exp Med. 2010;207:2817–2830. - PMC - PubMed
    1. Takahashi Y. Stem Cell Rep. 2018;10:314–328. - PMC - PubMed
    1. Takahashi Y. EBioMedicine. 2017;23:34–45. - PMC - PubMed

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