Self-Organizing, Symmetry Breaking, Isogenic Human Lung Buds on Microchips Identify Alveolar Stem Cells as Novel Targets of SARS-CoV-2 (preprint)

Although lung disease is the primary clinical outcome in COVID-19 patients, how SARS-CoV-2 induces lung pathology remains elusive. Here we describe a high-throughput platform to generate self-organizing, nearly identical, and genetically-matched human lung buds derived from hESCs cultured on micropatterned substrates. Synthetic lung buds resemble human fetal lungs and display proximo-distal patterning of alveolar and airway tissue directed by KGF. They are susceptible to infection by SARS-CoV-2 and endemic coronaviruses and can be used to track cell type-specific cytopathic effects in hundreds of lung buds in parallel. We detected an increased susceptibility to infection in alveolar cells and identified cycling alveolar stem cells as new targets of SARS-CoV-2. We used this platform to test neutralizing antibodies that efficiently blocked SARS-CoV-2 infection and transmission. Synthetic lung buds offer unlimited, rapid and scalable access to disease-relevant tissue that recapitulates human lung morphogenesis to identify key vulnerabilities for COVID-19 and respiratory viruses.Funding: This work was supported by the Pershing Square Foundation, NIH grants P01AI138398-S1, 2U19AI111825, and R01AI091707-10S1, a George Mason University Fast Grant, the BAWD Foundation, the G. Harold and Leila Y. Mathers Charitable Foundation, and private funding from the Rockefeller University.Conflict of Interest: A.H.B. is a co-founder of 2 startup companies: Rumi Scientific Inc. and OvaNova Laboratories, LLC and serves on their scientific advisory boards. C.M.R. is a founder of Apath LLC, a Scientific Advisory Board member of Imvaq Therapeutics, Vir Biotechnology, and Arbutus Biopharma, and an advisor for Regulus Therapeutics and Pfizer. All other authors declare no competing interests.

Self-organized stem cell-derived human lung buds with proximo-distal patterning and novel targets of SARS-CoV-2 (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic and the lack of therapeutics hinders pandemic control1-2. Although lung disease is the primary clinical outcome in COVID-19 patients1-3, how SARS-CoV-2 induces tissue pathology in the lung remains elusive. Here we describe a high-throughput platform to generate tens of thousands of self-organizing, nearly identical, and genetically matched human lung buds derived from human pluripotent stem cells (hPSCs) cultured on micropatterned substrates. Strikingly, in vitro-derived human lung buds resemble fetal human lung tissue and display in vivo-like proximo-distal coordination of alveolar and airway tissue differentiation whose 3D epithelial self-organization is directed by the levels of KGF. Single-cell transcriptomics unveiled the cellular identities of airway and alveolar tissue and the differentiation of WNThi cycling alveolar stem cells, a human-specific lung cell type4. These synthetic human lung buds are susceptible to infection by SARS-CoV-2 and endemic coronaviruses and can be used to track cell type-dependent susceptibilities to infection, intercellular transmission and cytopathology in airway and alveolar tissue in individual lung buds. Interestingly, we detected an increased susceptibility to infection in alveolar cells and identified cycling alveolar stem cells as targets of SARS-CoV-2. We used this platform to test neutralizing antibodies isolated from convalescent plasma that efficiently blocked SARS-CoV-2 infection and intercellular transmission. Our platform offers unlimited, rapid and scalable access to disease-relevant lung tissue that recapitulate key hallmarks of human lung development and can be used to track SARS-CoV-2 infection and identify candidate therapeutics for COVID-19.

TMEM41B is a pan-flavivirus host factor (preprint)

SUMMARY Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTS TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection. TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes. TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.