Influence of mesenchymal and biophysical components on distal lung organoid differentiation.

BACKGROUND: Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation. METHODS: Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC). RESULTS: We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation. CONCLUSION: Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.

Metabolomic profiling of human pluripotent stem cell differentiation into lung progenitors.

Metabolism is vital to cellular function and tissue homeostasis during human lung development. In utero, embryonic pluripotent stem cells undergo endodermal differentiation toward a lung progenitor cell fate that can be mimicked in vitro using induced human pluripotent stem cells (hiPSCs) to study genetic mutations. To identify differences between wild-type and surfactant protein B (SFTPB)-deficient cell lines during endoderm specification toward lung, we used an untargeted metabolomics approach to evaluate the developmental changes in metabolites. We found that the metabolites most enriched during the differentiation from pluripotent stem cell to lung progenitor cell, regardless of cell line, were sphingomyelins and phosphatidylcholines, two important lipid classes in lung development. The SFTPB mutation had no metabolic impact on early endodermal lung development. The identified metabolite signatures during lung progenitor cell differentiation may be utilized as biomarkers for normal embryonic lung development.

TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds.

The use of decellularized whole-organ scaffolds for bioengineering of organs is a promising avenue to circumvent the shortage of donor organs for transplantation. However, recellularization of acellular scaffolds from multicellular organs like the lung with a variety of different cell types remains a challenge. Multipotent cells could be an ideal cell source for recellularization. Here we investigated the hierarchical differentiation process of multipotent ES-derived endoderm cells into proximal airway epithelial cells on acellular lung scaffolds. The first cells to emerge on the scaffolds were TP63+ cells, followed by TP63+/KRT5+ basal cells, and finally multi-ciliated and secretory airway epithelial cells. TP63+/KRT5+ basal cells on the scaffolds simultaneously expressed KRT14, like basal cells involved in airway repair after injury. Removal of TP63 by CRISPR/Cas9 in the ES cells halted basal and airway cell differentiation on the scaffolds. These findings suggest that differentiation of ES-derived endoderm cells into airway cells on decellularized lung scaffolds proceeds via TP63+ basal cell progenitors and tracks a regenerative repair pathway. Understanding the process of differentiation is key for choosing the cell source for repopulation of a decellularized organ scaffold. Our data support the use of airway basal cells for repopulating the airway side of an acellular lung scaffold.

Limitations of recellularized biological scaffolds for human transplantation.

A shortage of donor organs for transplantation and the dependence of the recipients on immunosuppressive therapy have motivated researchers to consider alternative regenerative approaches. The answer may reside in acellular scaffolds generated from cadaveric human and animal tissues. Acellular scaffolds are expected to preserve the architectural and mechanical properties of the original organ, permitting cell attachment, growth, and differentiation. Although theoretically, the use of acellular scaffolds for transplantation should pose no threat to the recipient's immune system, experimental data have revealed significant immune responses to allogeneic and xenogeneic transplanted scaffolds. Herein, we review the various factors of the scaffold that could trigger an inflammatory and/or immune response, thereby compromising its use for human transplant therapy. In addition, we provide an overview of the major cell types that have been considered for recellularization of the scaffold and their potential contribution to triggering an immune response.

Conversion of human and mouse fibroblasts into lung-like epithelial cells.

Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.

Acid Sphingomyelinase Inhibition Attenuates Cell Death in Mechanically Ventilated Newborn Rat Lung.

RATIONALE: Premature infants subjected to mechanical ventilation (MV) are prone to lung injury that may result in bronchopulmonary dysplasia. MV causes epithelial cell death and halts alveolar development. The exact mechanism of MV-induced epithelial cell death is unknown. OBJECTIVES: To determine the contribution of autophagy to MV-induced epithelial cell death in newborn rat lungs. METHODS: Newborn rat lungs and fetal rat lung epithelial (FRLE) cells were exposed to MV and cyclic stretch, respectively, and were then analyzed by immunoblotting and mass spectrometry for autophagy, apoptosis, and bioactive sphingolipids. MEASUREMENTS AND MAIN RESULTS: Both MV and stretch first induce autophagy (ATG 5-12 [autophagy related 5-12] and LC3B-II [microtubule-associated proteins 1A/1B light chain 3B-II] formation) followed by extrinsic apoptosis (cleaved CASP8/3 [caspase-8/3] and PARP [poly(ADP-ribose) polymerase] formation). Stretch-induced apoptosis was attenuated by inhibiting autophagy. Coimmunoprecipitation revealed that stretch promoted an interaction between LC3B and the FAS (first apoptosis signal) cell death receptor in FRLE cells. Ceramide levels, in particular C16 ceramide, were rapidly elevated in response to ventilation and stretch, and C16 ceramide treatment of FRLE cells induced autophagy and apoptosis in a temporal pattern similar to that seen with MV and stretch. SMPD1 (sphingomyelin phosphodiesterase 1) was activated by ventilation and stretch, and its inhibition prevented ceramide production, LC3B-II formation, LC3B/first apoptosis signal interaction, caspase-3 activation, and, ultimately, FLRE cell death. SMPD1 inhibition also attenuated ventilation-induced autophagy and apoptosis in newborn rats. CONCLUSIONS: Ventilation-induced ceramides promote autophagy-mediated cell death, and identifies SMPD1 as a potential therapeutic target for the treatment of ventilation-induced lung injury in newborns.

CFTR rescue with VX-809 and VX-770 favors the repair of primary airway epithelial cell cultures from patients with class II mutations in the presence of Pseudomonas aeruginosa exoproducts.

BACKGROUND: Progressive airway damage due to bacterial infections, especially with Pseudomonas aeruginosa remains the first cause of morbidity and mortality in CF patients. Our previous work revealed a repair delay in CF airway epithelia compared to non-CF. This delay was partially prevented after CFTR correction (with VRT-325) in the absence of infection. Our goals were now to evaluate the effect of the Orkambi combination (CFTR VX-809 corrector + VX-770 potentiator) on the repair of CF primary airway epithelia, in infectious conditions. METHODS: Primary airway epithelial cell cultures from patients with class II mutations were mechanically injured and wound healing rates and transepithelial resistances were monitored after CFTR rescue, in the absence and presence of P. aeruginosa exoproducts. RESULTS: Our data revealed that combined treatment with VX-809 and VX-770 elicited a greater beneficial impact on airway epithelial repair than VX-809 alone, in the absence of infection. The treatment with Orkambi was effective not only in airway epithelial cell cultures from patients homozygous for the F508del mutation but also from heterozygous patients carrying F508del and another class II mutation (N1303 K, I507del). The stimulatory effect of the Orkambi treatment was prevented by CFTR inhibition with GlyH101. Finally, Orkambi combination elicited a slight but significant improvement in airway epithelial repair and transepithelial resistance, despite the presence of P. aeruginosa exoproducts. CONCLUSIONS: Our findings indicate that Orkambi may favor airway epithelial integrity in CF patients with class II mutations. Complementary approaches would however be needed to further improve CFTR rescue and airway epithelial repair.

Explant Culture for Studying Lung Development.

Lung development is a complex process that requires the input of various signaling pathways to coordinate the specification and differentiation of multiple cell types. Ex vivo culture of the lung is a very useful technique that represents an attractive model for investigating many different processes critical to lung development, function, and disease pathology. Ex vivo cultured lungs remain comparable to the in vivo lung both in structure and function, which makes them more suitable than cell cultures for physiological studies. Lung explant cultures offer several significant advantages for studies of morphogenetic events that guide lung development including budding, branching, and fusion. It also maintains the native physiological interactions between cells in the developing lung, enabling investigations of the direct and indirect signaling taking place between tissues and cells throughout the developmental process. Studying temporal and spatial control of gene expression by transcriptional factors using different reporters to understand their regulatory function at different moments of development is another valuable advantage of lung explants culture.

Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

Chronic Pseudomonas aeruginosa lung infections are associated with progressive epithelial damage and lung function decline. In addition to its role in tissue injury, the persistent presence of P. aeruginosa-secreted products may also affect epithelial repair ability, raising the need for new antivirulence therapies. The purpose of our study was to better understand the outcomes of P. aeruginosa exoproducts exposure on airway epithelial repair processes to identify a strategy to counteract their deleterious effect. We found that P. aeruginosa exoproducts significantly decreased wound healing, migration, and proliferation rates, and impaired the ability of directional migration of primary non-cystic fibrosis (CF) human airway epithelial cells. Impact of exoproducts was inhibited after mutations in P. aeruginosa genes that encoded for the quorum-sensing (QS) transcriptional regulator, LasR, and the elastase, LasB, whereas impact was restored by LasB induction in ΔlasR mutants. P. aeruginosa purified elastase also induced a significant decrease in non-CF epithelial repair, whereas protease inhibition with phosphoramidon prevented the effect of P. aeruginosa exoproducts. Furthermore, treatment of P. aeruginosa cultures with 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a QS inhibitor, abrogated the negative impact of P. aeruginosa exoproducts on airway epithelial repair. Finally, we confirmed our findings in human airway epithelial cells from patients with CF, a disease featuring P. aeruginosa chronic respiratory infection. These data demonstrate that secreted proteases under the control of the LasR QS system impair airway epithelial repair and that QS inhibitors could be of benefit to counteract the deleterious effect of P. aeruginosa in infected patients.-Ruffin, M., Bilodeau, C., Maillé, É., LaFayette, S. L., McKay, G. A., Trinh, N. T. N., Beaudoin, T., Desrosiers, M.-Y., Rousseau, S., Nguyen, D., Brochiero, E. Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

Deleterious impact of hyperglycemia on cystic fibrosis airway ion transport and epithelial repair.

BACKGROUND: Cystic fibrosis (CF)-related diabetes (CFRD) is associated with faster pulmonary function decline. Thus, we evaluated the impact of hyperglycemia on airway epithelial repair and transepithelial ion transport, which are critical in maintaining lung integrity and function. METHODS: Non-CF and CF airway epithelial cells were exposed to low (LG) or high (HG) glucose before ion current and wound repair rate measurements. RESULTS: CFTR and K+ currents decreased after HG treatments. HG also reduced the wound healing rates of non-CF and CF cell monolayers. Although CFTR correction with VRT-325 accelerated the healing rates of CF cells monolayers under LG conditions, this improvement was significantly abrogated under HG conditions. CONCLUSIONS: Our data highlights a deleterious impact of hyperglycemia on ion transport and epithelial repair functions, which could contribute to the deterioration in lung function in CFRD patients. HG may also interfere with the beneficial effects of CFTR rescue on airway epithelial repair.

Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells.

The epithelial response to bacterial airway infection, a common feature of lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis, has been extensively studied. However, its impact on cystic fibrosis transmembrane conductance regulator (CFTR) channel function is not clearly defined. Our aims were, therefore, to evaluate the effect of Pseudomonas aeruginosa on CFTR function and expression in non-cystic fibrosis airway epithelial cells, and to investigate its impact on ΔF508-CFTR rescue by the VRT-325 corrector in cystic fibrosis cells. CFTR expression/maturation was evaluated by immunoblotting and its function by short-circuit current measurements. A 24-h exposure to P. aeruginosa diffusible material (PsaDM) reduced CFTR currents as well as total and membrane protein expression of the wildtype (wt) CFTR protein in CFBE-wt cells. In CFBE-ΔF508 cells, PsaDM severely reduced CFTR maturation and current rescue induced by VRT-325. We also confirmed a deleterious impact of PsaDM on wt-CFTR currents in non-cystic fibrosis primary airway cells as well as on the rescue of ΔF508-CFTR function induced by VRT-325 in primary cystic fibrosis cells. These findings show that CFTR function could be impaired in non-cystic fibrosis patients infected by P. aeruginosa. Our data also suggest that CFTR corrector efficiency may be affected by infectious components, which should be taken into account in screening assays of correctors.

Regulation of normal and cystic fibrosis airway epithelial repair processes by TNF-α after injury.

Chronic infection and inflammation have been associated with progressive airway epithelial damage in patients with cystic fibrosis (CF). However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Our objective was to study the regulation of repair mechanisms by tumor necrosis factor-α (TNF-α), a major component of inflammation in CF, in a model of mechanical wounding, in two bronchial cell lines, non-CF NuLi and CF CuFi. We observed that TNF-α enhanced the NuLi and CuFi repair rates. Chronic exposure (24-48 h) to TNF-α augmented this stimulation as well as the migration rate during repair. The cellular mechanisms involved in this stimulation were then evaluated. First, we discerned that TNF-α induced metalloproteinase-9 release, epidermal growth factor (EGF) shedding, and subsequent EGF receptor transactivation. Second, TNF-α-induced stimulation of the NuLi and CuFi wound-closure rates was prevented by GM6001 (metalloproteinase inhibitor), EGF antibody (to titrate secreted EGF), and EGF receptor tyrosine kinase inhibitors. Furthermore, we recently reported a relationship between the EGF response and K(+) channel function, both controlling bronchial repair. We now show that TNF-α enhances KvLQT1 and K(ATP) currents, while their inhibition abolishes TNF-α-induced repair stimulation. These results indicate that the effect of TNF-α is mediated, at least in part, through EGF receptor transactivation and K(+) channel stimulation. In contrast, cell proliferation during repair was slowed by TNF-α, suggesting that TNF-α could exert contrasting actions on repair mechanisms of CF airway epithelia. Finally, the stimulatory effect of TNF-α on airway wound repair was confirmed on primary airway epithelial cells, from non-CF and CF patients.