Initially thought of as primarily a respiratory infection, SARS-CoV-2 is now implicated in substantial central nervous system (CNS) pathology3,4. CNS symptoms include ischemic strokes, hemorrhages, seizures, encephalopathy, encephalitis-meningitis, anosmia, post-infectious syndromes, and neuro-vasculopathy, collectively described in up to 85% of ICU patients5-8. Several reports appear to meet established criteria for infectious encephalitis9.
SARS-CoV-2 can utilize angiotensin-converting enzyme 2 (ACE2) as a receptor although other receptors have been proposed10,11. Recent studies on single cell RNA-seq (sc-RNA-seq) datasets indicate low levels of ACE2 expression in brain cells, however, expression is relatively high in some neurovascular unit (NVU) components, particularly in brain pericytes12-14. Autopsy series have suggested the potential for SARS-CoV-2 to spread throughout the brain, especially within vascular and immune cells. They note ischaemic brain lesions accompanied by widespread activation of astrocytes and cell death1,15. The potential for a SARS-CoV-2 elicited neurovasculopathy supports the development of new models to study tropism and pathology.
Brain pericytes are derived from neural crest stem cells (NCSCs), and are uniquely positioned in the neurovascular unit (NVU), physically linking endothelial and astrocytic cells16. Embedded within the basement membrane, pericytes connect, coordinate and regulate signals from neighboring cells to generate responses critical for CNS function in both healthy and disease states, including blood-brain barrier permeability, neuroinflammation, neuronal differentiation, and neurogenesis in the adult brain17-19.
We found that GFP+ pericyte-like cells (PLCs) generated in vitro from human pluripotent stem cell (hPSC)-derived NCSCs expressed the standard pericyte markers NG2 and PDGFR-b (Fig. 1a-b)20. We detected appreciable ACE2 mRNA and protein in 2D cultured PLCs compared with cultured human neural precursors (Extended Data Fig. 1a-d). To assess SARS-CoV-2 PLC tropism, we exposed PLCs to authentic SARS-CoV-2 at MOI 0.5, then harvested supernatant and cells daily (Fig. 1c). We found that the percentage of SARS-CoV-2 nucleocapsid (SNP)-positive cells and viral RNA as measured by qRT-PCR increased daily up to 72 hours post infection (h.p.i.), from 0% to 65% SNP positive, with viral RNA load increasing up to ~1000-fold (Fig. 1d-e). Plaque assay from supernatants on Vero E6 cells showed ~100-fold increased infectious virus production at 24 h.p.i with increased virus particles as well as viral titers compared to baseline, suggesting viral production by PLCs (Fig. 1f-g)21.
Demonstrating the infectability of PLCs led us to explore their effects on SARS-CoV-2 tropism within cortical brain organoids (COs). To this end, we developed an ‘assembloid’ in which GFP+ PLCs are integrated into mature COs. We thus generated PLC-containing cortical organoids (PCCOs), by seeding 2x105 GFP+ PLCs into wells containing COs at 60div (days in vitro) (Fig. 2a). By 74div, using tissue clearing and light sheet microscopy, we observed GFP+ cells integrating into COs as cell clusters, subsequently spreading across the surface and penetrating into the CTIP2+ cortical plate-like zone in COs (Fig. 2b-c).
We found that PCCOs maintained similar structural architecture and cellular compositions as traditional COs (Fig. 2d-f and Extended Data Fig. 2a-b). The GFP+ PLCs within PCCOs show GFP mRNA expression and retained standard pericyte markers expression (Extended Data Fig. 3a-b). However, we found that within PCCOs, PLCs attuned GFAP-positive astrocyte expression and morphology, which more closely resembled a classically described ‘star’-shape with end-feet like structures seen in mature astrocytes that were adjacent to PLCs (Fig. 2g)22,23. These results evidenced features of astrocytic maturation compared to traditional COs24. Moreover, we detected laminin-b1 protein adjacent to PLCs, suggesting accumulation of basement membrane (BM), which is normally absent from traditional COs (Fig. 2h-i). Confocal imaging confirmed localization of astrocytes and PLCs with laminin (Fig. 2j). Together, these results suggest PCCOs recapitulate the structural architecture of PLCs-BM-astrocytes described within the vertebral neurovascular unit (Fig. 2k)16.
We additionally characterized PCCOs by single cell RNA-seq (sc-RNA-seq). Compared to COs, PCCOs showed an ~23% shift from progenitor to neuronal populations, which was validated with TBR1 immunostaining (Fig. 2e, Extended Data Fig. 4a-d and Supplementary Dataset 1)25-27. Tandem mass spectrometry using isobaric labeling of PCCOs compared with COs supported these results, revealing that GFAP, TBR1, DCX, and STMN2 formed an upregulated protein module, suggesting an effect of PLCs on neuronal differentiation in PCCOs (Extended Data Fig. 5a-b, Supplementary Dataset 1).
We next exposed PCCOs to SARS-CoV-2 at MOI 0.5 for 72h (Fig. 3a). Compared with traditional COs that showed scant neuroglial cells positive for the established viral SNP protein as reported28,29, PCCOs showed a significantly higher proportion of SNP+ cells (10% vs. 1% in PCCOs vs. COs, p <0.0001, t-test, Fig. 3b-f, Extended Data Fig. 6a-b, Supplementary Dataset 2). qRT-PCR showed a corresponding ~50-fold increase in viral RNA in exposed PCCOs over COs (Fig. 3g, Supplementary Dataset 2).
We then compared the cellular infection vulnerability to SARS-CoV-2 in COs vs. PCCOs. In COs, we found <1% NeuN+/SNP+ cells or GFAP+/SNP+ cells and no discernable effect of viral exposure on CO characteristics (Extended Data Fig. 7a-e). In many ways, it seemed that the presence of SARS-CoV-2 was innocuous to COs. In contrast, in PCCOs we found the majority of SNP+ cells co-localized with GFP+ PLCs and surrounding GFAP+ astrocytes in virus-exposed PCCOs (Fig. 3e-e’ and h). Confocal imaging demonstrated that astrocytes were not only adjacent to the infected PLCs but were themselves SNP+ (Fig. 3i). To transcriptionally profile cellular constituents, we performed sc-RNA-seq at 72h.p.i. We detected SARS-CoV-2 reads in ~2% of cells in PCCOs, overwhelmingly confined to astrocytes, but not neurons (Fig. 3j-m). There were no detectable SARS-CoV-2 reads in infected COs (Supplementary Dataset 1). These data suggest that infection of astrocytes is mediated by the presence of the PLCs population.
Finally, to explore pathogenesis of SARS-CoV-2 in PCCOs, we performed immunostaining and observed a significant increase (~20%) in percent of cells evidencing programmed cell death (cleaved caspase 3 and p53-positive) in infected PCCOs (Fig. 4a-b, Supplementary Dataset 2). sc-RNA-seq indicated that the source of the cell death was largely confined to astrocytes, consistent with the described selective vulnerability (Fig. 4c)30. Gene ontology (GO) term analysis of differentially expressed genes (DEGs) specific to astrocytes highlighted inflammatory and genotoxic stress activation (Fig. 4d). This correlated with activation of type I interferon transcriptional response, and with upregulation of IFIT1, IFI44, ISG15 in virus exposed compared to mock infected PCCOs (Fig. 4e, Supplementary Dataset 3)31. Several type I interferons signaling cascade genes (STAT1, STAT2) and an ISG15-effector gene (USP18) were also upregulated (Fig. 4f, Supplementary Dataset 2)32. Increased expression of ISG15 was confirmed by qRT-PCR in infected PCCOs (Fig. 4g). These results implicate astrocytic pathology in SARS-CoV-2 inflammatory brain pathology, mediated through the type I interferon pathway.
Here we demonstrate that PLCs can be productively infected by SARS-CoV-2 and through integration of PLCs into COs, we established a novel PCCO ‘assembloid’. Within PCCOs, we found PLCs establish characteristics of PLCs-BM-astrocytes structure and increase the cellular proportion of neuronal population, mimicking reported functions of human brain pericytes in vivo. Upon exposure to SARS-CoV-2, we observed robust infection within PCCOs and consequent induction of neuronal death and type I interferon responses. Furthermore, we demonstrate that PLCs can serve as viral ‘replication hubs’, supporting the viral invasion and spread to other cell types, including astrocytes.
Although SARS-CoV-2 invasion into human CNS has been modeled in 3D brain organoids, choroid plexus (ChP) organoids, and K18-hACE2 transgenic mice, evidence suggests that most neural cells have little to no capacity for SARS-CoV-2 infection. On the other hand, the presence of any cells expressing ACE2 or other receptors may be sufficient to initiate infection28,29,33,34, motivating further work to understand the receptor expression profile and impact on infection at the human NVU in vivo. Drawing on clinical and experimental data supporting potential vascular entry and ACE2 expression in pericytes, our PCCO SARS-CoV-2 infection model presents an alternative route to infection. The PCCO model could be further improved by incorporating other NVU-component cell types, which might lend itself to other uses35. Our work provides a powerful model to study SARS-CoV-2, and may be useful to model other infectious diseases.