The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of ...pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5
basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5
cells in basal organoids revealed a distinct population of ITGA6
ITGB4
mitotic cells, whose offspring further segregated into a TNFRSF12A
subfraction that comprised about ten per cent of KRT5
basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.
Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory ...sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%–50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%–50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.
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•Cas9 RNP and AAV can be used to efficiently gene edit human airway basal stem cells•This method yields >30% allelic correction without selection markers or antibiotics•Correction of >30% ΔF508 alleles restores CFTR function to near non-CF levels•Corrected stem cells can differentiate after embedding in the scaffold for engraftment
Vaidyanathan et al. use Cas9 and AAV to correct the CF causing ΔF508 mutation in >30% of alleles in airway basal stem cells from CF patients and embed corrected cells on a scaffold for engraftment. This method restores physiologic CFTR function and provides an ex vivo strategy to treat cystic fibrosis.
Wnt5a-Ror signaling is a conserved pathway that regulates morphogenetic processes during vertebrate development R. T. Moon
,
119, 97-111 (1993); I. Oishi
,
8, 645-654 (2003), but its downstream ...signaling events remain poorly understood. Through a large-scale proteomic screen in mouse embryonic fibroblasts, we identified the E3 ubiquitin ligase Pdzrn3 as a regulatory target of the Wnt5a-Ror pathway. Upon pathway activation, Pdzrn3 is degraded in a β-catenin-independent, ubiquitin-proteasome system-dependent manner. We developed a flow cytometry-based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 stability. Epistatically, Pdzrn3 is regulated independently of Kif26b, another Wnt5a-Ror effector. Wnt5a-dependent degradation of Pdzrn3 requires phosphorylation of three conserved amino acids within its C-terminal LNX3H domain M. Flynn, O. Saha, P. Young,
11, 235 (2011), which acts as a bona fide Wnt5a-responsive element. Importantly, this phospho-dependent degradation is essential for Wnt5a-Ror modulation of cell migration. Collectively, this work establishes a Wnt5a-Ror cell morphogenetic cascade involving Pdzrn3 phosphorylation and degradation.
Noncanonical WNT pathways function independently of the β-catenin transcriptional co-activator to regulate diverse morphogenetic and pathogenic processes. Recent studies showed that noncanonical ...WNTs, such as WNT5A, can signal the degradation of several downstream effectors, thereby modulating these effectors' cellular activities. The protein domain(s) that mediates the WNT5A-dependent degradation response, however, has not been identified. By coupling protein mutagenesis experiments with a flow cytometry-based degradation reporter assay, we have defined a protein domain in the kinesin superfamily protein KIF26B that is essential for WNT5A-dependent degradation. We found that a human disease-causing KIF26B mutation located at a conserved amino acid within this domain compromises the ability of WNT5A to induce KIF26B degradation. Using pharmacological perturbation, we further uncovered a role of glycogen synthase kinase 3 (GSK3) in WNT5A regulation of KIF26B degradation. Lastly, based on the identification of the WNT5A-responsive domain, we developed a new reporter system that allows for efficient profiling of WNT5A-KIF26B signaling activity in both somatic and stem cells. In conclusion, our study identifies a new protein domain that mediates WNT5A-dependent degradation of KIF26B and provides a new tool for functional characterization of noncanonical WNT5A signaling in cells.
Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror ...pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/β-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.
Aging organisms lose the ability to induce stress responses, becoming vulnerable to protein toxicity and tissue damage. Neurons can signal to peripheral tissues to induce protective ...organelle-specific stress responses. Recent work shows that glia can independently induce such responses. Here, we show that overexpression of heat shock factor 1 (
) in the four astrocyte-like cephalic sheath cells of
induces a non-cell-autonomous cytosolic unfolded protein response, also known as the heat shock response (HSR). These animals have increased lifespan and heat stress resistance and decreased protein aggregation. Glial HSR regulation is independent of canonical thermosensory circuitry and known neurotransmitters but requires the small clear vesicle release protein UNC-13. HSF-1 and the FOXO transcription factor DAF-16 are partially required in peripheral tissues for non-cell-autonomous HSR, longevity, and thermotolerance. Cephalic sheath glial
overexpression also leads to pathogen resistance, suggesting a role for this signaling pathway in immune function.
•A new, broadly applicable system for determining bacterial drug resistance in space.•Potential measures to improve the management of urinary tract infections that can afflict astronauts.•Determining ...if bacteria respond to microgravity as a stress akin to other insults.
Human immune response is compromised and bacteria can become more antibiotic resistant in space microgravity (MG). We report that under low-shear modeled microgravity (LSMMG), stationary-phase uropathogenic Escherichia coli (UPEC) become more resistant to gentamicin (Gm), and that this increase is dependent on the presence of σs (a transcription regulator encoded by the rpoS gene). UPEC causes urinary tract infections (UTIs), reported to afflict astronauts; Gm is a standard treatment, so these findings could impact astronaut health. Because LSMMG findings can differ from MG, we report preparations to examine UPEC's Gm sensitivity during spaceflight using the E. coli Anti-Microbial Satellite (EcAMSat) as a free-flying “nanosatellite” in low Earth orbit. Within EcAMSat's payload, a 48-microwell fluidic card contains and supports study of bacterial cultures at constant temperature; optical absorbance changes in cell suspensions are made at three wavelengths for each microwell and a fluid-delivery system provides growth medium and predefined Gm concentrations. Performance characterization is reported here for spaceflight prototypes of this payload system. Using conventional microtiter plates, we show that Alamar Blue (AB) absorbance changes can assess the Gm effect on E. coli viability, permitting telemetric transfer of the spaceflight data to Earth. Laboratory results using payload prototypes are consistent with wellplate and flask findings of differential sensitivity of UPEC and its ∆rpoS strain to Gm. if σs plays the same role in space MG as in LSMMG and Earth gravity, countermeasures discovered in recent Earth studies (aimed at weakening the UPEC antioxidant defense) to control UPEC infections would prove useful also in space flights. Further, EcAMSat results should clarify inconsistencies from previous space experiments on bacterial antibiotic sensitivity and other issues.
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