The influence of SARS-CoV-2 spike protein exposure on retinal development in the human retinal organoids

Abstract

Background: Pregnant women are considered a high-risk population for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as the virus can infect the placenta and embryos. Recently, SARS-CoV-2 has been widely reported to cause retinal pathological changes and to infect the embryonic retina. The infection of host cells by SARS-CoV-2 is primarily mediated through spike (S) protein, which also plays a crucial role in the pathogenesis of SARS-CoV-2. However, it remains poorly understood how the S protein of SARS-CoV-2 affects retinal development, and the underlying mechanism has not yet been clarified.

Methods: We used human embryonic stem cell-derived retinal organoids (hEROs) as a model to study the effect of S protein exposure at different stages of retinal development. hEROs were treated with 2 μg/mL of S protein on days 90 and 280. Immunofluorescence staining, RNA sequencing, and RT-PCR were performed to assess the influence of S protein exposure on retinal development at both early and late stages.

Results: The results showed that ACE2 and TMPRSS2, the receptors facilitating SARS-CoV-2 entry into host cells, were expressed in hEROs. Exposure to the S protein induced an inflammatory response in both the early and late stages of retinal development in the hEROs. Additionally, RNA sequencing indicated that early exposure of the S protein to hEROs affected nuclear components and lipid metabolism, while late-stages exposure resulted in changes to cell membrane components and the extracellular matrix.

Conclusion: This work highlights the differential effects of SARS-CoV-2 S protein exposure on retinal development at both early and late stages, providing insights into the cellular and molecular mechanisms underlying SARS-CoV-2-induced developmental impairments in the human retina.

Keywords: Retinal development; Retinal organoid; SARS-CoV-2; Spike protein.

Fig. 1

Generation and identification of hEROs in vitro. A Schematic of the hEROs self-organization protocol. B Bright-field view showing the representative images of hEROs at days 18, 60, 90, 150, and 280. Scale bar, 100 μm. C Immunostaining of hEROs at days 18, 60, 90, 150, and 280 for CHX10⁺, Vimentin (Vim)⁺, HuC/D⁺, CRX⁺, and Recoverin (RECV)⁺ cells. Scale bar, 50 μm

Fig. 2

Expression of SARS-CoV-2 receptor in hEROs during embryonic retinal development. A The relative mRNA expression of ACE2 and TMPRSS2 (TMP) in hECSs (ES) and hEROs (D60, D90, D150, and D280). B Representative images of the expression of ACE2⁺ and TMP⁺ cells at day 60, 90, 150, and 280 by immunofluorescence staining. Scale bar, 50 μm. Data are presented as means ± SD. ***P < 0.001, ****P < 0.0001

Fig. 3

The influence of the SARS-CoV-2 S protein exposure on the development of hEROs. A Schematic of S protein treatment of hEROs at different points. B The relative mRNA levels of ACE2 and TMP in hEROs at day 90 after S protein exposure, compared to control groups . C The relative mRNA levels of ACE2 and TMP in hEROs at day 280 after S protein exposure, compared to control groups . D Representative images of the expression of RECV⁺/ACE2⁺ cells and CRX⁺/ACE2⁺ cells in hEROs at days 90 and 280 after S protein exposure. E The ratio of CRX-positive cells/ area (μm²) and the average fluorescence intensity of RECV- positive cells in 90-day hEROs after S protein exposure, compared to control groups . F The ratio of CRX-positive cells/ area (μm²) and the average fluorescence intensity of RECV- positive cells in 280-day hEROs after S protein exposure, compared to control groups. Data are presented as means ± SD. Scale bar, 50 μm

Fig. 4

RNA sequencing analysis of the influence of S protein exposure on hEROs on days 90 and 280. A Schematic of the RNA sequencing protocol for S protein treatment of hEROs. B The hierarchical clustering heatmap of the control and S protein treatment groups on day 90. C The hierarchical clustering heatmap of the control and S protein treatment groups on day 280. D The hierarchical clustering heatmap of genes associated with photoreceptors, retinal ganglion cells, and progenitor cells in hEROs on days 90 and 280 for both groups, respectively. E The hierarchical clustering heatmap showed the expression of the SARS-CoV-2 receptor ACE2 and TMPRSS2 in hEROs on days 90 and 280 for both groups, respectively

Fig. 5

S protein exposure exhibits different effects on retinal development at different stages. A Enriched GO terms in CC category in  90-day hEROs after S protein exposure, compared to control groups. The statistically significant enriched GO terms were ranked by gene numbers. B Enriched GO terms in CCs category in 280-day hEROs after S protein exposure, compared to control groups. The statistically significant enriched GO terms were ranked by gene numbers. C Enriched GO terms in MF category in 90-day hEROs after S protein exposure, compared to control groups. The statistically significant enriched GO terms were ranked by gene numbers. D Enriched GO terms in MF category in 280-day hEROs after S protein exposure, compared to control groups. The statistically significant enriched GO terms were ranked by gene numbers. E Enriched GO terms in BP category in  90-day hEROs after S protein exposure, compared to control groups. The statistically significant enriched GO terms were ranked by gene numbers. F Enriched GO terms in BP category in 280-day hEROs after S protein exposure, compared to control groups. The statistically significant enriched GO terms were ranked by gene numbers

Fig. 6

S protein exposure disrupts retinal development at different stages. A The enrichment of KEGG pathways in 90-day hEROs after S protein exposure, compared to control groups. The statistically significant enriched KEGG pathways were ranked by gene numbers. B The enrichment of KEGG pathways in 280-day hEROs after S protein exposure, compared to control groups. The statistically significant enriched KEGG pathways were ranked by gene numbers. C, D GSEA analysis shows the enrichment of gene sets associated with cytokine receptor interaction and oxidative phosphorylation in 90-day hEROs after S protein exposure, compared to control groups. E, F GSEA analysis shows the enrichment of gene sets associated with endocytosis and ubiquitin mediated proteolysis in 280-day hEROs after S protein exposure, compared to control groups

Fig. 7

S protein exposure shows the influences of retinal development at the molecular levels. A The  KEGG pathway signaling interaction network of the DEGs in 90-day hEROs after S protein exposure, compared to control groups. B The KEGG pathway signaling interaction network of the DEGs in 280-day hEROs after S protein exposure, compared to control groups. C Volcano plot shows the DEGs of S protein exposure in hEROs at day 90, compared to control groups. D Volcano plot shows the DEGs of S protein exposure in hEROs at day 280, compared to control groups. E, F The relative mRNA expression level of HK2, LDHA, MYD88, and TLR4 in 90-day hEROs  after S protein exposure, compared to control groups. G, H The relative mRNA expression level of COL1A1, COL6A3, ID3, and TGFβ1 in 280-day hEROs  after S protein exposure, compared to control groups. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001