Airway Basal Stem Cells in COVID-19 Exhibit a Proinflammatory Signature and Impaired Mucocililary Differentiation.

Airway basal stem cells (BSCs) play a critical role in epithelial regeneration. Whether coronavirus disease (COVID-19) affects BSC function is unknown. Here, we derived BSC lines from patients with COVID-19 using tracheal aspirates (TAs) to circumvent the biosafety concerns of live-cell derivation. We show that BSCs derived from the TAs of control patients are bona fide bronchial BSCs. TA BSCs from patients with COVID-19 tested negative for severe acute respiratory syndrome coronavirus 2 RNA; however, these so-termed COVID-19-exposed BSCs in vitro resemble a predominant BSC subpopulation uniquely present in patients with COVID-19, manifested by a proinflammatory gene signature and STAT3 hyperactivation. Furthermore, the sustained STAT3 hyperactivation drives goblet cell differentiation of COVID-19-exposed BSCs in an air-liquid interface. Last, these phenotypes of COVID-19-exposed BSCs can be induced in control BSCs by cytokine cocktail pretreatment. Taken together, acute inflammation in COVID-19 exerts a long-term impact on mucociliary differentiation of BSCs.

Rho/SMAD/mTOR triple inhibition enables long-term expansion of human neonatal tracheal aspirate-derived airway basal cell-like cells.

BACKGROUND: Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture. METHODS: We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates. RESULTS: Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium. CONCLUSIONS: Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease. IMPACT: Airway epithelial basal cell-like cell lines were derived from human neonatal tracheal aspirates. mTOR inhibition significantly extends in vitro proliferation of neonatal tracheal aspirate-derived basal cell-like cells (nTAD BCCs). nTAD BCCs can be differentiated into functional airway epithelium. nTAD BCCs provide a novel model to investigate perinatal lung development and diseases.

Engineering pulmonary vasculature in decellularized rat and human lungs.

Bioengineered lungs produced from patient-derived cells may one day provide an alternative to donor lungs for transplantation therapy. Here we report the regeneration of functional pulmonary vasculature by repopulating the vascular compartment of decellularized rat and human lung scaffolds with human cells, including endothelial and perivascular cells derived from induced pluripotent stem cells. We describe improved methods for delivering cells into the lung scaffold and for maturing newly formed endothelium through co-seeding of endothelial and perivascular cells and a two-phase culture protocol. Using these methods we achieved ∼75% endothelial coverage in the rat lung scaffold relative to that of native lung. The regenerated endothelium showed reduced vascular resistance and improved barrier function over the course of in vitro culture and remained patent for 3 days after orthotopic transplantation in rats. Finally, we scaled our approach to the human lung lobe and achieved efficient cell delivery, maintenance of cell viability and establishment of perfusable vascular lumens.

Engineered composite tissue as a bioartificial limb graft.

The loss of an extremity is a disastrous injury with tremendous impact on a patient's life. Current mechanical prostheses are technically highly sophisticated, but only partially replace physiologic function and aesthetic appearance. As a biologic alternative, approximately 70 patients have undergone allogeneic hand transplantation to date worldwide. While outcomes are favorable, risks and side effects of transplantation and long-term immunosuppression pose a significant ethical dilemma. An autologous, bio-artificial graft based on native extracellular matrix and patient derived cells could be produced on demand and would not require immunosuppression after transplantation. To create such a graft, we decellularized rat and primate forearms by detergent perfusion and yielded acellular scaffolds with preserved composite architecture. We then repopulated muscle and vasculature with cells of appropriate phenotypes, and matured the composite tissue in a perfusion bioreactor under electrical stimulation in vitro. After confirmation of composite tissue formation, we transplanted the resulting bio-composite grafts to confirm perfusion in vivo.

Notch signaling modulates MUC16 biosynthesis in an in vitro model of human corneal and conjunctival epithelial cell differentiation.

PURPOSE: Notch proteins are a family of transmembrane receptors that coordinate binary cell fate decisions and differentiation in wet-surfaced epithelia. We sought to determine whether Notch signaling contributes to maintaining mucosal homeostasis by modulating the biosynthesis of cell surface-associated mucins in an in vitro model of human corneal (HCLE) and conjunctival (HCjE) epithelial cell differentiation. METHODS: HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction. RESULTS: Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4. CONCLUSIONS: These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface.