Nie J. et al. Validation and Establishment of the pseudovirus neutralization assay for SARS-CoV-2. SARS-CoV-2-entry pathogen (1104 PFU), luciferase was primarily recognized in Nurr1: GFP+ DA neurons as indicated by immunofluorescence staining (Fig. 1c). This shows that hPSC-derived DA neurons are permissive to SARS-CoV-2 disease when subjected to the pathogen disease. b, Consultant confocal picture of DA neuron xenograft stained with antibodies against Nurr1-GFP and ACE2. Scale pub=50m. c, Consultant confocal picture of DA neuron xenograft at 24 hpi stained with antibodies against Nurr1-GFP and Luc. Scale pub=60m. d. qRT-PCR evaluation of total RNA extracted from hPSC-derived DA neurons at 48 hpi of SARS-CoV-2 disease (MOI=0.2) for viral N sgRNA. The graph depicts the mean sgRNA level normalized to ideals had been determined by unpaired two-tailed College students t check. ** 0.01. Next, hPSC-derived DA neurons had been contaminated with SARS-CoV-2 (USA-WA1/2020, MOI=0.2). At 48 hours (??)-BI-D post disease (hpi), qRT-PCR evaluation using primers focusing on subgenomic N transcripts verified that quite a lot of (??)-BI-D viral replication could possibly be detected in the RNA level in contaminated hPSC-derived DA neurons (Fig. 1d). Immunostaining for the SARS-N proteins confirmed solid SARS-CoV-2 disease of DA neurons (Fig. 1e). Finally, transmitting electron microscopy was utilized to detect the current presence of viral contaminants in SARS-CoV-2 contaminated hPSC-derived DA neurons (Fig. 1f). General, these and tests confirm that human being hPSC-derived DA neurons are permissive to SARS-CoV-2 and support productive infection. RNA-seq analysis was applied to compare mock-infected or SARS-CoV-2 infected hPSC-derived DA neurons. Robust viral infection was detected in SARS-CoV-2 infected DA neurons (Fig. 1g). Moreover, plotting these datasets by principal component analysis (PCA, Fig. 1h) and clustering analysis (Fig. 1i) demonstrated that the infected DA neurons occupied a distinct transcriptional space compared to mock-infected DA neurons. In contrast, no obvious transcriptional changes were observed following SARS-CoV-2 exposure of hPSC-derived cortical neurons. In hPSC-derived DA neurons, we next analyzed DA neuron marker expression and found that levels of and were decreased in SARS-CoV-2 infected samples (Fig. 1j). In particular, markers of the A9 DA neurons ? the subtype of ventral midbrain DA neurons most affected in PD ? such as expression following SARS-CoV-2 infection, indicating an increased vulnerability of human DA neurons expressing A9 subtype specific markers to SARS-CoV-2 infection (Fig. 1k, ?,1l1l). Volcano plots and heatmap of SARS-CoV-2 infected versus mock-infected hPSC-derived DA neurons showed robust induction of chemokine and cytokine transcripts, including (Fig. 2a, ?,2b).2b). Those transcriptional changes were specific to DA neuron cultures and again not observed in cortical neuron cultures, in line with our previous work showing a lack of susceptibility of cortical neurons to SARS-CoV-27. In infected DA neurons, inflammation-associated genes were also upregulated in SARS-CoV-2 infected DA neurons (Fig. 2c). Ingenuity Pathway Analysis highlighted the senescence pathway as the top regulated pathway in SARS-CoV-2 infected DA neurons (Fig. 2d), a finding further corroborated by the upregulated expression of key genes involved in the senescence CD63 pathway (Fig. 2e). Beta-galactosidase (Beta-Gal), a biomarker of cellular senescence8, was also upregulated in SARS-CoV-2 infected hPSC-derived DA neurons (Fig. 2f, ?,2g).2g). qRT-PCR analysis was performed for examining the expression of senescence-pathway associated genes, including and was significantly upregulated in SARS-CoV-2 infected DA neurons while was significantly downregulated (Fig. 2h). Finally, transmission electron microscopy detected lipofuscin in SARS-CoV-2 infected DA neurons as an additional senescence-associated marker of DA neurons15 (Fig. 2i). The induction of DA neuron senescence and evidence of increased vulnerability of human A9 DA neurons suggest that SARS-CoV-2 infection could serve as a potential degenerative trigger for DA neurons. Open in a separate window Figure 2. SARS-CoV-2 infection induces senescence of DA neurons.a, Volcano plot indicating (??)-BI-D differentially expressed genes in mock or SARS-CoV-2 infected hPSC-derived DA neurons at 48 hpi (MOI=0.2). Differentially expressed genes (p-adjusted value.