Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract.

Organ fibrosis is a lethal outcome of autoimmune rheumatic diseases such as systemic sclerosis. Myofibroblasts are scar-forming cells that are ultimately responsible for the excessive synthesis, deposition and remodelling of extracellular matrix proteins in fibrosis. Advances have been made in our understanding of the mechanisms that keep myofibroblasts in an activated state and control myofibroblast functions. However, the mechanisms that help myofibroblasts to persist in fibrotic tissues remain poorly understood. Myofibroblasts evade apoptosis by activating molecular mechanisms in response to pro-survival biomechanical and growth factor signals from the fibrotic microenvironment, which can ultimately lead to the acquisition of a senescent phenotype. Growing evidence suggests that myofibroblasts and senescent myofibroblasts, rather than being resistant to apoptosis, are actually primed for apoptosis owing to concomitant activation of cell death signalling pathways; these cells are poised to apoptose when survival pathways are inhibited. This knowledge of apoptotic priming has paved the way for new therapies that trigger apoptosis in myofibroblasts by blocking pro-survival mechanisms, target senescent myofibroblast for apoptosis or promote the reprogramming of myofibroblasts into scar-resolving cells. These novel strategies are not only poised to prevent progressive tissue scarring, but also have the potential to reverse established fibrosis and to regenerate chronically injured tissues.

Evasion of apoptosis by myofibroblasts: a hallmark of fibrotic diseases.

Dear Editor, Coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 13 million confirmed cases and more than 580,045 deaths across 218 countries and geographical regions as of July 16, 2020. This novel coronavirus primarily causes respiratory illness with clinical manifestations largely resembling those of SARS. However, neurological symptoms including headache, anosmia, ageusia, confusion, seizure, and encephalopathy have also been frequently reported in COVID-19 patients. In a study of 214 hospitalized COVID-19 patients in Wuhan, China, neurologic findings were reported in 36.4% of patients, and were more commonly observed in patients with severe infections (45.5%). Similarly, a study from France reported neurologic findings in 84.5% (49/58) of COVID-19 patients admitted to hospital. Importantly, a recent study in Germany demonstrated that SARS-CoV-2 RNA could be detected in brain biopsies in 36.4% (8/22) of fatal COVID-19 cases, which highlights the potential for viral infections in the human brain. To date, there has been no direct experimental evidence of SARS-CoV-2 infection in the human central nervous system (CNS). We recently demonstrated that SARS-CoV-2 could infect and replicate in cells of neuronal origin. In line with this finding, we showed that SARS-CoV-2 could infect and damage the olfactory sensory neurons of hamsters. In addition, angiotensin-converting enzyme 2 (ACE2), the entry receptor of SARS-CoV-2, is widely detected in the brain and is highly concentrated in a number of locations including substantia nigra, middle temporal gyrus, and posterior cingulate cortex. Together, these findings suggest that the human brain might be an extra-pulmonary target of SARS-CoV-2 infection. To explore the direct involvement of SARS-CoV-2 in the CNS in physiologically relevant models, we assessed SARS-CoV-2 infection in induced pluripotent stem cells (iPSCs)-derived human neural progenitor cells (hNPCs), neurospheres, and brain organoids. We first evaluated the expression of ACE2 and key coronavirus entry-associated proteases in hNPCs. Our data suggested that ACE2, TMPRSS2, cathepsin L, and furin were readily detected in the hNPCs (Supplementary information, Fig. S1). Next, we challenged iPSC-derived hNPCs with SARSCoV-2 at 10 multiplicity of infection (MOI) and with SARS-CoV as a control. Supernatant was harvested at 0, 24, and 48 h post infection (hpi) for virus replication assessment. Interestingly, our data suggested that SARS-CoV-2, but not SARS-CoV, could replicate in hNPCs (Fig. 1a; Supplementary information, Fig. S2). In addition, we quantified the cell viability of SARS-CoV-2infected hNPCs. Importantly, SARS-CoV-2 infection significantly reduced the viability of hNPCs to 4.7% (P < 0.0001) and 2.5% (P < 0.0001) of that of the mock-infected hNPCs at 72 and 120 hpi, respectively (Fig. 1a). In contrast to the substantial cytotoxicity induced by SARS-CoV-2 in the infected hNPCs, SARS-CoV-2 infection did not significantly upregulate interferon (Supplementary information, Fig. S3) and pro-inflammatory (Supplementary information, Fig. S4) response in the infected hNPCs. Next, we challenged 3D neurospheres with SARS-CoV-2 and harvested supernatant samples from the infected neurospheres at 0, 24, 48, and 72 hpi for virus replication assessment. We found the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) copy number significantly increased in a time-dependent manner (Fig. 1b, left). In addition, a significant amount of infectious virus particles were released from the infected neurospheres as determined by plaque assays (Fig. 1b, right). In parallel, SARS-CoV-2-infected neurospheres were cryosectioned and immunostained for viral antigen assessment. Importantly, SARS-CoV-2 nucleocapsid (N) protein was readily detected across the infected neurospheres but no positive signals were detected in the mock-infected neurospheres (Fig. 1c). Furthermore, electron microscopy detected extensive viral particles in vacuoles within the double-membrane structures, which may represent sites of viral particle formation (Fig. 1d). These findings indicate that neurospheres were permissive to SARS-CoV-2 infection and supported productive virus replication. Next, we examined whether SARS-CoV-2 could infect 3D human brain organoids. We generated iPSC-derived human brain organoids using previously described protocols. The 35-day-old brain organoids showed self-organizing internal morphology with fluid-filled ventricular-like structures resembling that of developing cerebral cortex (Fig. 1e). Cryosectioning and immunostaining were performed to determine the expression and distribution of neuronal markers in 35-day-old brain organoids. Pan-neurons, early forebrain, and hNPCs markers were identified by TUJ1, PAX6, and NESTIN staining, respectively. The TUJ1 staining identified a primitive cortical plate with early neurons (Fig. 1e), whereas PAX6 staining represented the radial glia in the cerebral cortex (Fig. 1e). In addition, NESTIN staining identified active proliferating NPCs in the brain organoids (Fig. 1e). Overall, these results indicated that telencephalon development and cerebral neurogenesis could be modelled by our organoid system. To investigate whether the brain organoids were permissive to SARS-CoV-2 infection, human iPSC-derived 35-day-old brain organoids were challenged with SARS-CoV-2. Importantly, extensive SARS-CoV-2 antigen was detected in the infected samples at 72 hpi (Fig. 1f), indicating that SARS-CoV-2 directly infected the brain organoids. Immunofluorescence staining and confocal microscopy revealed SARS-CoV-2-N signals in the peripheral regions (Fig. 1f, arrows) and in deeper regions of the brain organoids (Fig. 1f, white arrowheads). In addition, cell-cell fusion was readily detected in regions with robust SARS-CoV-2 infection (Fig. 1f, yellow arrowheads). No SARS-CoV-2-N signals were detected in the mock-infected brain organoids (Fig. 1f). We next analyzed supernatant samples from infected brain organoids to evaluate SARS-CoV-2 virus particle release. The results demonstrated SARS-CoV-2 RdRp gene copy number increased in a timedependent manner, suggesting active release of progeny virus particles from infected brain organoids (Fig. 1g, left). Specifically,

/pdf/sars-cov-2-infects-human-neural-progenitor-cells-and-brain-4c9bg306p4.pdf

SARS-CoV-2 infects human neural progenitor cells and brain organoids.

Getting to the Cores of Autism

Multi-lineage Human iPSC-Derived Platforms for Disease Modeling and Drug Discovery

Complex Oscillatory Waves Emerging from Cortical Organoids Model Early Human Brain Network Development.

Pdgfra-expressing (Pdgfra+) cells have been implicated as progenitors in many mesenchymal tissues. To determine lineage potential, we generated PdgfrartTA knockin mice using CRISPR/Cas9. During lung maturation, counter to a prior study reporting that Pdgfra+ cells give rise equally to myofibroblasts and lipofibroblasts, lineage tracing using PdgfrartTA;tetO-cre mice indicated that ~95% of the lineaged cells are myofibroblasts. Genetic ablation of Pdgfra+ cells using PdgfrartTA-driven diphtheria toxin (DTA) led to alveolar simplification, demonstrating that these cells are essential for building the gas exchange surface area. In the adult bleomycin model of lung fibrosis, lineaged cells increased to contribute to pathological myofibroblasts. In contrast, in a neonatal hyperoxia model of bronchopulmonary dysplasia (BPD), lineaged cells decreased and do not substantially contribute to pathological myofibroblasts. Our findings revealed complexity in the behavior of the Pdgfra-lineaged cells as exemplified by their distinct contributions to myofibroblasts in normal maturation, BPD and adult fibrosis.

Jing Gu

Papers

Complex Oscillatory Waves Emerging from Cortical Organoids Model Early Human Brain Network Development.

Pdgfra marks a cellular lineage with distinct contributions to myofibroblasts in lung maturation and injury response.