Human motor neurons derived from induced pluripotent stem cells are susceptible to SARS-CoV-2 infection

Abstract

Introduction: COVID-19 typically causes Q7 respiratory disorders, but a high proportion of patients also reports neurological and neuromuscular symptoms during and after SARSCoV-2 infection. Despite a number of studies documenting SARS-CoV-2 infection of various neuronal cell populations, the impact of SARS-CoV-2 exposure on motor neuronal cells specifically has not been investigated so far.

Methods: Thus, by using human iPSC-derived motor neurons (iPSC-MNs) we assessed: (i) the expression of SARS-CoV-2 main receptors; (ii) iPSC-MN infectability by SARS-CoV-2; and (iii) the effect of SARS-CoV-2 exposure on iPSC-MN transcriptome.

Results: Gene expression profiling and immunofluorescence (IF) analysis of the main host cell receptors recognized by SARS-CoV-2 revealed that all of them are expressed in iPSC-MNs, with CD147 and NRP1 being the most represented ones. By analyzing SARS-CoV-2 N1 and N2 gene expression over time, we observed that human iPSC-MNs were productively infected by SARS-CoV-2 in the absence of cytopathic effect. Supernatants collected from SARS-CoV-2-infected iPSC-MNs were able to re-infect VeroE6 cells. Image analyses of SARS-CoV-2 nucleocapsid proteins by IF confirmed iPSC-MN infectability. Furthermore, SARS-CoV-2 infection in iPSCMNs significantly altered the expression of genes (IL-6, ANG, S1PR1, BCL2, BAX, Casp8, HLA-A, ERAP1, CD147, MX1) associated with cell survival and metabolism, as well as antiviral and inflammatory response.

Discussion: These results suggest for the very first time that SARS-CoV-2 can productively infect human iPSC-derived MNs probably by binding CD147 and NRP1 receptors. Such information will be important to unveil the biological bases of neuromuscular disorders characterizing SARS-CoV-2 infection and the so called long-COVID symptoms.

Keywords: COVID-19; SARS-CoV-2 infection; iPSC-derived motor neurons; long-COVID; neuroinflammation; neuromuscular disorders.

FIGURE 1

Graphical representation of the study workflow. SARS-CoV-2 host receptor gene expression was assessed on iPSC-derived motor neurons (iPSC-MNs) by RT-qPCR and immunofluorescence. iPSC-MN were then in vitro infected by SARS-CoV-2 and viral infection/replication was assessed by a multidisciplinary approach using RT-qPCR, TCID₅₀, immunofluorescence and quantigene Plex techniques. The effect of viral infection on iPSC-MN homeostasis was determined by analyzing the alteration of their transcriptome.

FIGURE 2

Expression of SARS-CoV-2 human receptors on iPSC-MNs and A549-hACE2 cells. (A) Representative images of differentiated iPSC-MNs obtained from 3 healthy control individuals. Expression of neuronal (βIII-Tubulin, red and green, and SMI-312, green) and motoneuronal (HB9, red and ChAT, green) markers is shown in merged images. Nuclei were stained with DAPI (blue). Bar, 10 μm. (B) Gene expression analyses of ACE2, CD147, NRP1, TMPRSS2, and FURIN in A549-hACE2 cells and iPSC- MNs by Real-time qPCR. Results are presented as mean ± SEM; n ≥ 4 for each cell line/iPSC-MN; the Student’s t-test was used with the p-value threshold of 0.05. Significance is indicated as follows: **p < 0.01; ***p < 0.001. Representative immunofluorescence images for CD147 and NRP1 markers (green) in iPSC-MNs and in A549-hACE2 cells are shown in panels (C,D), respectively. The expression of ACE2 is shown in red (C) and in green (D). Nuclei were stained with DAPI (blue). The neuronal marker SMI-312 (red) is shown only in (C). Bars correspond to 20 μm in both (C,D).

FIGURE 3

Assessment of viral replication in iPSC-MNs. (A) Upon in vitro challenge of iPSC-MNs with 1 MOI of SARS-CoV-2, the infection was monitored at 6, 24, 48, and 72 hpi. Results correspond to the absolute viral copy number/μl of the SARS-CoV-2 N1 and N2 target sequences from cell supernatants that were quantified through a single-step Real-time qPCR by referring to a standard curve for Ct values (IDT, Coralville, IA, USA). Results are presented as mean ± SEM from ≥4 independent replicates each on iPSC-MNs derived from the 3 enrolled healthy subjects (Supplementary Table 1). (B) TCID₅₀ analyses on VeroE6 cell infectability after exposure to iPSC-MN-infected supernatants, specifically the undiluted virus (I) and five serial dilutions (1:3), at 24, 48, and 72 hpi. Viral infection was assessed by cytopathic effect on VeroE6 cells as represented by colored well. The plate is representative of a single experiment which was performed once in quadruplicate (x, y, z, w) for each of the three different iPSC-MN lines. (C) Titration of SARS-CoV-2 virus in VeroE6 supernatants at 24, 48, and 72 hpi from data shown in (B). Data are shown as TCID₅₀. Results are presented as mean ± SEM from four independent replicates each on iPSC-MNs derived from the three enrolled healthy subjects.

FIGURE 4

Assessment of viral replication in iPSC-MNs. (A) Expression of SARS-CoV-2 viral genes ORF7A, ORF3A, ORF8, RDRP, S, E, and N by QuantiGene Plex Gene expression technology in uninfected (Mock) and SARS-CoV-2-infected iPSC-MNs. Results shown on the heatmap correspond to the mean ± SEM from ≥1 independent replicates on each iPSC-MNs derived from 2 healthy control subjects (Supplementary Table 1). (B) Real-time qPCR expression analyses of N1, S1, S2, and E2 viral genes in uninfected (Mock) and SARS-CoV-2-infected iPSC-MNs. Results are presented as mean ± SEM from ≥4 independent replicates on iPSC-MNs derived from the 3 healthy control subjects (Supplementary Table 1). (C) Representative immunofluorescence images of N protein (red) and neuronal SMI-312 marker (green) in Mock- and SARS-CoV-2-infected iPSC-MNs at 48 hpi. Nuclei were stained with DAPI (blue). Bars correspond to 20 μm. (D) Representative magnified immunofluorescence images and 3D reconstruction for N protein and SMI-312 marker in SARS-CoV-2-infected iPSC-MNs at 48 hpi.

FIGURE 5

Gene expression analyses in SARS-CoV-2-infected iPSC-MNs. (A) Heatmap representation of Real-time qPCR expression data of 46 genes in uninfected (Mock) and SARS-CoV-2-infected iPSC-MNs at 72 hpi. (B). Expression analyses of the statistically significant genes in SARS-CoV-2-infected vs. uninfected (Mock) iPSC-MNs at 72 hpi are shown in (A). mRNA quantification was performed by Real-time qPCR and calculated by the 2–ΔΔCt equation. Results in (A,B) correspond to the mean ± SEM from ≥4 independent replicates on each iPSC-MN derived from the three enrolled healthy subjects (Supplementary Table 1); the Student’s t-test was used with the p-value threshold of 0.05. Significance is indicated as follows: *p < 0.05.