Expression of Endogenous Angiotensin-Converting Enzyme 2 in Human Induced Pluripotent Stem Cell-Derived Retinal Organoids

Abstract

Angiotensin-converting enzyme 2 (ACE2) was identified as the main host cell receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its subsequent infection. In some coronavirus disease 2019 (COVID-19) patients, it has been reported that the nervous tissues and the eyes were also affected. However, evidence supporting that the retina is a target tissue for SARS-CoV-2 infection is still lacking. This present study aimed to investigate whether ACE2 expression plays a role in human retinal neurons during SARS-CoV-2 infection. Human induced pluripotent stem cell (hiPSC)-derived retinal organoids and monolayer cultures derived from dissociated retinal organoids were generated. To validate the potential entry of SARS-CoV-2 infection in the retina, we showed that hiPSC-derived retinal organoids and monolayer cultures endogenously express ACE2 and transmembrane serine protease 2 (TMPRSS2) on the mRNA level. Immunofluorescence staining confirmed the protein expression of ACE2 and TMPRSS2 in retinal organoids and monolayer cultures. Furthermore, using the SARS-CoV-2 pseudovirus spike protein with GFP expression system, we found that retinal organoids and monolayer cultures can potentially be infected by the SARS-CoV-2 pseudovirus. Collectively, our findings highlighted the potential of iPSC-derived retinal organoids as the models for ACE2 receptor-based SARS-CoV-2 infection.

Keywords: ACE2; COVID-19; SARS-CoV-2; SARS-CoV-2 pseudovirus; induced pluripotent stem cells; organoids; spike protein.

Figure 1

Establishment of human induced pluripotent stem cell (iPSC)-derived retinal organoids and monolayer cultures. (A) Schematic showing the examination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity in the human iPSC-derived retinal organoids. (B) Schematic illustrating the generation of human iPSC-derived retinal organoids from human peripheral blood mononuclear cells. (C) Schematic diagram illustrating the differentiation protocol for human induced pluripotent stem cell (hiPSC)-derived retinal organoids. (D) Bright-field representative images of each stage of the retinal organoid differentiation. Scale bar, 200 µm. (E) Immunofluorescence showing the putative retinal markers of day 150 human iPSC-derived organoids. HuC/HuD: neuron marker; cone-rod homeobox (CRX), rhodopsin (Rho), and recoverin [32]: photoreceptor markers. Scale bar, 50 µm. (F) Bright-field images showing the representative morphology of day 150 retinal organoids with a dense translucent projection at the apical edge (arrowhead). The double arrow shows the outer nuclear layer. Scale bar, 50 µm. (G) Bright-field images of monolayer cultures derived from dissociated retinal organoids. Scale bar, 50 µm. (H) Immunofluorescence indicated that the monolayer cultures were positively stained for the CRX (green) and recoverin (red) photoreceptor markers. Scale bar, 50 µm.

Figure 2

Analysis of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) expressions on human iPSC-derived retinal organoids and monolayer cultures. (A) qPCR showing the ACE2 and TMPRSS2 gene expressions in retinal organoids (Day 60, Day 150, and Day 200); monolayer cultures; retinal ganglion cells (RGCs); and pigmented epithelium cells (RPEs). The expression of the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used for normalization. * p < 0.05, ** p < 0.01, and *** p < 0.001, and error bars are the standard deviation. (B) Immunofluorescence analysis of monolayer cultures with anti-ACE2. ACE2 expression was analyzed in monolayer cultures and stained in green at the cell membrane. Nuclei was stained positive for DAPI (blue). Scale bar, 50 µm. (C) The expression of TMPRSS2 (green) in the monolayer cultures. Cell nuclei was stained positive for DAPI. (D) The expression and localization of ACE2 and TMPRSS2 in Day-150 retinal organoids were evaluated by an immunofluorescence analysis. The area with ACE2 expression was stained in red and, with TMPRSS2 expression, stained in green. Nuclei were stained positive for DAPI. Right subpanel represents the boxed region of the image in the left subpanel with high magnification.

Figure 3

SARS-CoV-2 pseudovirus infection in retinal organoids and monolayer cultures. (A) 293T cells were transfected with a plasmid encoding a lentiviral backbone expressing a GFP fluorescent protein, a plasmid-expressing spike protein, and the other necessary proteins essential for virion formation. The transfected cells produced lentiviral particles with a spike protein on their surfaces, and these viral particles were able to infect cells that express the ACE2 receptor. (B) Infection of monolayer cultures with the SARS-CoV-2 pseudovirus was performed in uninfected and the SARS-CoV-2 pseudovirus at MOI 0.1, 0.5, and 1 at 3 and 6 days post-infection (dpi). Results represent the mean ± SD from three independent experiments. The presence of a SARS-CoV-2 pseudovirus is shown in green and nuclei in blue. (C) The graph shows the fold-change of the SARS-CoV-2 pseudovirus genome copies of uninfected and the SARS-CoV-2 pseudovirus-infected monolayer cultures at the indicated time points: 3 dpi and 6 dpi. Representative data from three independent experiments are shown. ** p < 0.01, and *** p < 0.001. (D) RT-PCR analysis of GFP expression in monolayer cultures infected with different MOI (0.1, 0.5, and 1) and at two time points: 3 dpi and 6 dpi. (E) Representative bright-field images of day-150 retinal organoids (left subpanel) infected by the SARS-CoV-2 pseudovirus. The GFP signals (right subpanel) indicate the infection of the SARS-CoV-2 pseudovirus (MOI = 1, dpi = 6). Scale bar, 50 µm. (F) Immunofluorescence staining showing ACE2 expression localization at the apical edge of the retinal organoids. (G,H) qPCR and RT-PCR analyses of GFP expression in the SARS-CoV-2 pseudovirus-infected retinal organoids at 3 and 6 dpi. ** p < 0.01, and *** p < 0.001.

Figure 4

RNA-Seq analysis of hiPSC-derived retinal organoids and monolayer cultures after SARS-CoV-2 pseudovirus infection. (A) Volcano plot highlighting the transcript differentially expressed gene during the SARS-CoV-2 pseudovirus infection compared to the parental/uninfected. Red color defines an upregulated expression with a log₂ (fold change) > 2, and blue defines a downregulated expression with a log₂ (fold change) < 2. (B) Enriched gene oncology (GO) terms of differentially expressed genes in the cells. (C) Heatmap expression patterns of parental/uninfected compared to the infected SARS-CoV-2 pseudovirus retinal organoids and monolayer cultures. Red and blue colors indicate differentially expressed genes that were up- (red) and downregulated (blue). PE-S represents the represent the monolayer culture infection of the SARS-CoV-2 pseudovirus, and SE-S1-2 represents the retinal organoid infection of the SARS-CoV-2 pseudovirus. PE-C1 and SE-C1-2 represent the parental/uninfected control for the monolayer cultures and retinal organoids, respectively.

Figure 5

Overview of the SARS-CoV-2 infection in hiPSC-derived retinal organoids and monolayer cultures. General schematic of the experimental workflow of hiPSCs-derived retinal organoids and monolayer cultures. After the in vitro differentiation to the organoids or dissociation into the monolayer cultures, the organoids/cells can be used for disease modeling and therapeutic testing applications.