There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host–viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air–liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway.

Efforts to decipher chronic lung disease and to reconstitute functional lung tissue through regenerative medicine have been hampered by an incomplete understanding of cell-cell interactions governing tissue homeostasis. Because the structure of mammalian lungs is highly conserved at the histologic level, we hypothesized that there are evolutionarily conserved homeostatic mechanisms that keep the fine architecture of the lung in balance. We have leveraged single-cell RNA sequencing techniques to identify conserved patterns of cell-cell cross-talk in adult mammalian lungs, analyzing mouse, rat, pig, and human pulmonary tissues. Specific stereotyped functional roles for each cell type in the distal lung are observed, with alveolar type I cells having a major role in the regulation of tissue homeostasis. This paper provides a systems-level portrait of signaling between alveolar cell populations. These methods may be applicable to other organs, providing a roadmap for identifying key pathways governing pathophysiology and informing regenerative efforts.

Microglia play a role in the emergence and preservation of a healthy brain microenvironment. Dysfunction of microglia has been associated with neurodevelopmental and neurodegenerative disorders. Investigating the function of human microglia in health and disease has been challenging due to the limited models of the human brain available. Here, we develop a method to generate functional microglia in human cortical organoids (hCOs) from human embryonic stem cells (hESCs). We apply this system to study the role of microglia during inflammation induced by amyloid-β (Aβ). The overexpression of the myeloid-specific transcription factor PU.1 generates microglia-like cells in hCOs, producing mhCOs (microglia-containing hCOs), that we engraft in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement and chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by Aβ. Furthermore, in mhCOs, we observed reduced expression of Aβ-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer’s disease (AD) stage III. Finally, we assess the function of AD-associated genes highly expressed in microglia in response to Aβ using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. In summary, we provide a protocol to generate mhCOs that can be used in fundamental and translational studies as a model to investigate the role of microglia in neurodevelopmental and neurodegenerative disorders.

Single-cell RNA-sequencing data has revolutionized our ability to understand of the patterns of cell–cell and ligand–receptor connectivity that influence the function of tissues and organs. However, the quantification and visualization of these patterns in a way that informs tissue biology are major computational and epistemological challenges. Here, we present Connectome, a software package for R which facilitates rapid calculation and interactive exploration of cell–cell signaling network topologies contained in single-cell RNA-sequencing data. Connectome can be used with any reference set of known ligand–receptor mechanisms. It has built-in functionality to facilitate differential and comparative connectomics, in which signaling networks are compared between tissue systems. Connectome focuses on computational and graphical tools designed to analyze and explore cell–cell connectivity patterns across disparate single-cell datasets and reveal biologic insight. We present approaches to quantify focused network topologies and discuss some of the biologic theory leading to their design.

Heart failure is the leading cause of death in the United States and rapidly becoming the leading cause of death worldwide. While pharmacological treatments can reduce progression to heart failure following myocardial infarction, there still exists a need for new therapies that promote better healing post injury for a more functional cardiac repair and methods to understand how the changes to tissue mechanical properties influence cell phenotype and function following injury. To address this need, we have optimized a silk-based hydrogel platform containing cardiac tissue-derived extracellular matrix (cECM). These silk-cECM hydrogels have tunable mechanical properties, as well as rate-controllable hydrogel stiffening over time. In vitro, silk-cECM scaffolds led to enhanced cardiac fibroblast (CF) cell growth and viability with culture time. cECM incorporation improved expression of integrin an focal adhesion proteins, suggesting that CFs were able to interact with the cECM in the hydrogel. Subcutaneous injection of silk hydrogels in rats demonstrated that addition of the cECM led to endogenous cell infiltration and promoted endothelial cell ingrowth after 4 weeks in vivo. This naturally derived silk fibroin platform is applicable to the development of more physiologically relevant constructs that replicate healthy and diseased tissue in vitro and has the potential to be used as an injectable therapeutic for cardiac repair.