Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses

Abstract

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

Keywords: Hendra virus; Nipah virus; artery; biosafety-level-4 virology; developmental biology; endothelial cells; human pluripotent stem cells; vein.

Figure 1:. Efficient generation of human primitive streak and lateral mesoderm from hPSCs within 1 and 2 days, respectively

A) Artery and vein development B) Generating MIXL1⁺ mid primitive streak within 24 hours of hPSC differentiation (Loh et al., 2016), assayed by flow cytometry of MIXL1-GFP hPSCs (Davis et al., 2008) C) VEGF and BMP specify, whereas TGFβ and WNT repress, day 2 lateral mesoderm. Day 1 hPSC-derived primitive streak was treated with the indicated signals (concentrations indicated, and 10 nM Axitinib; 200 ng/mL NOGGIN; 1 μM DMH1, XAV939 and C59; 2 μM SB505124 and SB431542; base conditions contained 10 ng/mL BMP4 and/or 100 ng/mL VEGF) for 24 hours; qPCR was performed on day 2. See also Figure S1 and Table S5

Figure 2:. Efficient generation of human artery endothelial cells from hPSCs within 3 days

A) Summary of present work B) TGFβ promotes, while PI3K inhibits, day 3 artery formation. Day 2 hPSC-derived lateral mesoderm was treated with the indicated signals, including TGFβ agonist (Activin, 5–25 ng/mL), TGFβ inhibitor (SB505124, 2 μM) or PI3K inhibitor (GDC0941, 0.5–2 μM), for 24 hours; qPCR was performed on day 2 (base media: GDC0941+VEGF+XAV939+AA2P+TTNPB [left] and VEGF+XAV939+AA2P+TTNPB+Activin [right]). Gene expression was normalized to the base media lacking TGFβ or PI3K modulators. C) Immunostaining of H1 hPSCs either before or after differentiation into day 3 artery ECs; scale = 100 μm D) Flow cytometry of SOX17–2A-mPlum hPSCs before or after differentiation into day 3 artery ECs E) H1 and SUN004.1.9 hPSC lines differentiated using the present artery EC induction method or 4 prevailing EC differentiation protocols (Lian et al., 2014; Patsch et al., 2015; Sriram et al., 2015; Zhang et al., 2017); flow cytometry was performed on days 0, 3, 5 and 6 of differentiation; n.d. = not determined F) scRNAseq of H1 hPSCs differentiated towards artery ECs; the entire day 3 cell population was harvested without pre-selecting ECs; gene expression in log_e UMI units G) scRNAseq comparison of hPSC-derived day 3 artery ECs vs. Carnegie Stage 13 (CS13) human fetal artery ECs (Zeng et al., 2019); 6000 variable genes (top, each dot = 1 gene) or selected genes (bottom) shown See also Figure S2 and Tables S1 and S5

Figure 3:. Efficient generation of human vein endothelial cells from hPSCs within 4 days

A) Summary of present work B) Dually inhibiting TGFβ and NOTCH promotes day 3 vein gene expression. Day 2 hPSC-derived lateral mesoderm was further differentiated for 24 hours with the indicated signals, including TGFβ inhibitor (SB505124, 2 μM) and/or NOTCH inhibitor (RO4929097, 1 μM); qPCR performed on day 3; *P<0.05; **P<0.01 C) VEGF/ERK activation followed by inhibition is critical for vein formation. Day 2 hPSC-derived lateral mesoderm was further differentiated for 48 hours in the presence or absence of ERK inhibitor (PD0325901, 100 nM) for the indicated durations; qPCR performed on day 4 (base media: VEGF+SB505124+RO4929097+XAV939+DMH1+AA2P for days 3–4) D) Flow cytometry of H1 NR2F2–2A-GFP hPSCs before or after differentiation into day 5 vein ECs E) NR2F2 and VE-CADHERIN immunostaining of H1 hPSC-derived day 4 vein ECs; scale = 100 μm F) scRNAseq of H1 hPSCs differentiated towards vein ECs; the entire day 4 cell population was harvested without pre-selecting ECs; gene expression in log_e UMI units G) scRNAseq comparison of hPSC-derived day 4 vein ECs vs. Carnegie Stage 13 (CS13) human fetal vein ECs (Zeng et al., 2019); 6000 variable genes (top, each dot = 1 gene) or selected genes (bottom) shown See also Figure S3 and Tables S2 and S5

Figure 4:. Transcriptional and functional differences between hPSC-derived artery and vein endothelial cells

A) Bulk-population RNA-seq of H1 hPSC-derived day 3 artery vs. day 4 vein ECs (CD144⁺ FACS sorted), each dot = 1 gene B) 3D vascular networks formed by H1 hPSC-derived artery and vein ECs cocultured with fibroblasts in fibrin gel for 1 week (Kurokawa et al., 2017); scale = 200 μm C-E) Subcutaneous transplantation of CAG-AkaLuciferase-tdTomato⁺ SUN003.1 hPSC-derived artery and vein ECs, monitored by intravital imaging (to assess cell proliferation) and microscopy (4 weeks post-transplant); scale = 150 μm F) Flow cytometry of fluorescently-labelled, acetylated LDL uptake by H1 hPSC-derived artery and vein ECs within 4 hours G) SUN004.1.9 hPSC-derived artery and vein ECs exposed to shear stress or static conditions for 24 hours align to the direction of fluid flow, quantified by the angle of the long axis of CD144⁺ cell membranes; scale = 100 μm H) Beads coated with CAG-GFP⁺ SUN004.1.9 hPSC-derived vein ECs preferentially form vascular sprouts after embedding in 3D fibrin gel for 1 day, as opposed to artery EC-coated beads; scale = 100 μm I) H1 hPSC-derived artery and vein ECs were expanded for 5 days, and then treated with TNFα (10 ng/mL) or left untreated for 4 hours, followed by qPCR to assess TNFα-induced upregulation of immune cell adhesion molecules; **P<0.01 J) H1 hPSC-derived artery and vein ECs were expanded for 6 days, and then were treated with TNFα (10 ng/mL) or left untreated for 4 hours, prior to addition of fluorescent THP1 cells for 30 minutes; scale = 100 μm; *P<0.05 K) In Transwell assay, CAG-GFP⁺ SUN004.1.9 hPSC-derived vein ECs preferentially migrate towards Apelin-13; images of migrated cells situated on the bottom membrane surface; scale = 100 μm; *P<0.05 See also Figure S4 and Tables S3 and S5

Figure 5:. Nipah and Hendra viruses target artery cells

A) Syncytia (arrows) in H1 hPSC-derived artery and vein ECs inoculated with Nipah or Hendra viruses for 24 hours; scale = 25 μm B) Numbers of nuclei per cell after inoculation with Nipah or Hendra viruses for 24 hours (top); syncytia frequency (≥3 nuclei/cell; bottom) C) Live imaging of cocultured SUN004.1.9 GFP⁺ and H1 wild-type artery ECs infected with Nipah virus, tracking cell fusion into GFP⁺ syncytia, followed by death (scale = 200 μm, timestamp: hours:minutes) D) Live imaging showed Nipah- and Hendra-infected artery ECs displayed significantly greater median velocity (a measure of cell fusion) than vein ECs E) Fusion and GFP diffusion between 3 Nipah-infected artery ECs, followed by aggregation of their nuclei (scale bar = 20 μm, timestamp: hours:minutes) See also Figure S5 and Movie S1

Figure 6:. Artery cells express higher levels of EFNB2, the Nipah and Hendra virus receptor

A) Nipah and Hendra viral RNA replication in H1 hPSC-derived artery and vein ECs, as quantified by qPCR of culture media; *P<0.05; **P<0.01 B) scRNAseq of human adult lung ECs (Travaglini et al., 2020) C) Higher EFNB2 levels in hPSC-derived artery ECs relative to vein ECs, as shown by i) scRNAseq of day 3 artery vs. day 4 vein ECs and ii) flow cytometry for surface EFNB2 protein expression on artery vs. vein ECs, 4 days post-expansion D) Nipah or Hendra viral RNA replication in H1 and EFNB2^−/− hPSC-derived artery and vein ECs, as assayed by qPCR of culture media E) H1 and EFNB2^−/− hPSC-derived artery and vein ECs inoculated with GFP-encoding Nipah pseudovirus (Palomares et al., 2013); **P<0.01 See also Figure S6 and Table S5

Figure 7:. Nipah and Hendra viruses elicit minimal transcriptional changes in artery endothelial cells, despite extensive viral replication and cell fusion

A) Percentage of hPSC-derived artery EC transcriptome comprising viral reads, as quantified by RNA-seq; averaged from H1 and SUN004.1.9 hPSCs B) Transcriptome-wide differences between infected vs. uninfected artery ECs by RNA-seq; averaged from H1 and SUN004.1.9 hPSCs C,D) RNA-seq of infected hPSC-derived artery ECs, including viral read percentage in transcriptome; averaged from H1 and SUN004.1.9 hPSCs E) IFNβ secretion by hPSC-derived artery ECs; averaged from H1, SUN004.1.9 and SUN004.2 hPSCs; *P<0.05; n.s. = not significant See also Figure S7 and Table S4