Embryonic Mouse Lung Research Articles

Effects of PEGylated Fe–Fe3O4 core-shell nanoparticles on NIH3T3 and A549 cell lines

Magnetic nanoparticles are key components in many fields of science and industry. Especially in cancer diagnosis and therapy, they are involved in targeted drug delivery and hyperthermia applications due to their ability to be controlled remotely. In this study, a PEG-coated Fe/Fe3O4 core-shell nanoparticle with an average size of 20 nm and 13 nm and high room temperature coercivity (350 Oe) has been successfully synthesized. These nanoparticles were further tested for their effect on cellular toxicity (IC50) and proliferation by WST assay. In addition, their potential as anti-cancer agents were assessed using scratch assay in NIH3T3 mouse embryonic fibroblast and A549 non-small cell lung cancer cell lines.In previous reports, the IC50 values of the magnetite nanoparticles are reported at concentrations of 100 μg/ml and higher. In this study, IC50 value is observed to be at 1 μg/ml, which is significantly lower when compared to similar studies. In scratch assay, the Fe/Fe3O4 core-shell nanoparticle showed a higher inhibitory potential on cell motility in A549 lung cancer cells in comparison to the NIH3T3 cells mouse embryonic fibroblasts. This could be due to the accelerated release of free Fe ion from the Fe core, resulting in cell death. Consequently, data obtained from this study suggest that the synthesized nanoparticles can be a potential drug candidate with anti-cancer activity for chemotherapeutic treatment.

LKB1-AMPK Axis Negatively Regulates Ferroptosis by Inhibiting Fatty Acid Synthesis

Ferroptosis is a form of regulated necrotic cell death characterized by an iron-dependent lipid peroxidation. Although mounting evidence has demonstrated that diverse cellular metabolic processes contribute to ferroptosis, the detail mechanism by which cellular metabolism regulates ferroptosis is largely unknown. Here, we demonstrated that AMP-activated protein kinase (AMPK), a highly conversed master regulator of cellular energy homostasis is a crucial negative regulator of ferroptosis. Activation of AMPK by glucose starvation or a pharmaceutical activator suppresses ferroptotic cell death, and genetic knocking out of AMPK sensitizes cells to ferroptosis. Mechanistically, ferroptosis inducers activate AMPK which in turn phosphorylates acetyl-CoA carboxylase 1 (ACC1), leading to inhibition of fatty acid synthesis, lipid peroxide accumulation and ferroptosis. Importantly, loss of function of tumor suppressor liver kinase B1 (LKB1) sensitizes mouse embryonic fibroblasts (MEFs) and human non-small cell lung carcinoma cell lines to ferroptosis. Collectively, our results demonstrate the vital role of LKB1-AMPK-ACC1-FAS axis in dictating ferroptotic cell death, and suggest that malignant mutations in LKB1-AMPK signalling could predict the responsiveness of cancer cells to future ferroptosis-inducing therapies.

Mesenchyme-specific deletion of Tgf-β1 in the embryonic lung disrupts branching morphogenesis and induces lung hypoplasia

Proper lung development depends on the precise temporal and spatial expression of several morphogenic factors, including Fgf10, Fgf9, Shh, Bmp4, and Tgf-β. Over- or under-expression of these molecules often leads to aberrant embryonic or postnatal lung development. Herein, we deleted the Tgf-β1 gene specifically within the lung embryonic mesenchymal compartment at specific gestational stages to determine the contribution of this cytokine to lung development. Mutant embryos developed severe lung hypoplasia and died at birth due to the inability to breathe. Despite the markedly reduced lung size, proliferation and differentiation of the lung epithelium was not affected by the lack of mesenchymal expression of the Tgf-β1 gene, while apoptosis was significantly increased in the mutant lung parenchyma. Lack of mesenchymal expression of the Tgf-β1 gene was also associated with reduced lung branching morphogenesis, with accompanying inhibition of the local FGF10 signaling pathway as well as abnormal development of the vascular system. To shed light on the mechanism of lung hypoplasia, we quantified the phosphorylation of 226 proteins in the mutant E12.5 lung compared with control. We identified five proteins, Hrs, Vav2, c-Kit, the regulatory subunit of Pi3k (P85), and Fgfr1, that were over- or under-phosphorylated in the mutant lung, suggesting that they could be indispensable effectors of the TGF-β signaling program during embryonic lung development. In conclusion, we have uncovered novel roles of the mesenchyme-specific Tgf-β1 ligand in embryonic mouse lung development and generated a mouse model that may prove helpful to identify some of the key pathogenic mechanisms underlying lung hypoplasia in humans.

Autophagy and mTOR pathways in mouse embryonic stem cell, lung cancer and somatic fibroblast cell lines.

Embryonic developmental stages and regulations have always been one of the most intriguing aspects of science. Since the cancer stem cell discovery, striking for cancer development and recurrence, embryonic stem cells and control mechanisms, as well as cancer cells and cancer stem cell control mechanisms become important research materials. It is necessary to reveal the similarities and differences between somatic and cancer cells which are formed of embryonic stem cells divisions and determinations. For this purpose, mouse embryonic stem cells (mESCs), mouse skin fibroblast cells (MSFs) and mouse lung squamous cancer cells (SqLCCs) were grown in vitro and the differences between these three cell lines signalling regulations of mechanistic target of rapamycin (mTOR) and autophagic pathways were demonstrated by immunofluorescence and real-time polymerase chain reaction. Expressional differences were clearly shown between embryonic, cancer and somatic cells that mESCs displayed higher expressional level of Atg10, Hdac1 and Cln3 which are related with autophagic regulation and Hsp4, Prkca, Rhoa and ribosomal S6 genes related with mTOR activity. LC3 and mTOR protein levels were lower in mESCs than MSFs. Thus, the mechanisms of embryonic stem cell regulation results in the formation of somatic tissues whereas that these cells may be the causative agents of cancer in any deterioration.

Characterization of Tg(Etv4-GFP) and Etv5 RFP Reporter Lines in the Context of Fibroblast Growth Factor 10 Signaling During Mouse Embryonic Lung Development.

Members of the PEA3 transcription factors are emerging as bone fide targets for fibroblast growth factor (FGF) signaling. Among them, ETV4 and ETV5 appear to mediate FGF10 signaling during early embryonic lung development. In this paper, recently obtained Tg(Etv4-GFP) and Etv5CreERT2−RFP fluorescent reporter lines were generally characterized during early embryonic development and in the context of FGF10 signaling, in particular. We found that both Tg(Etv4-GFP) and Etv5CreERT2−RFP were primarily expressed in the epithelium of the lung during embryonic development. However, the expression of Etv5CreERT2−RFP was much higher than that of Tg(Etv4-GFP), and continued to increase during development, whereas Tg(Etv4-GFP) decreased. The expression patterns of the surrogate fluorescent protein GFP and RFP for ETV4 and ETV5, respectively, agreed with known regions of FGF10 signaling in various developing organs, including the lung, where ETV4-GFP was seen primarily in the distal epithelium and to a lesser extent in the surrounding mesenchyme. As expected, ETV5-RFP was restricted to the lung epithelium, showing a decreasing expression pattern from distal buds to proximal conducting airways. FGF10 inhibition experiments confirmed that both Etv4 and Etv5 are downstream of FGF10 signaling. Finally, we also validated that both fluorescent reporters responded to FGF10 inhibition in vitro. In conclusion, these two reporter lines appear to be promising tools to monitor FGF10/FGFR2b signaling in early lung development. These tools will have to be further validated at later stages and in other organs of interest.

HIV-Nef Protein Persists in the Lungs of Aviremic Patients with HIV and Induces Endothelial Cell Death.

It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte-activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV+ patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial-cadherin+ endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.

Alveolus-like organoid from isolated tip epithelium of embryonic mouse lung

Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such as HopX and Aquaporin5, and type 2 cell markers, such as Lamp3 and Surfactant apoproteins (Sftp) C and D. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.

Imaging and Analysis of Mouse Embryonic Whole Lung, Isolated Tissue, and Lineage-Labelled Cell Culture.

Research on lung development and disease frequently utilizes mouse models to conduct in vitro experiments. Such experiments involve multiple methodologically distinct stages, from careful consideration of mouse models used to obtain biological samples, to the culturing and imaging of those samples, and finally, to post-imaging analysis. Here, we detail basic protocols to assist with each of these stages. First, we discuss harvesting and preparing biological samples; second, we focus on culturing embryonic whole lung explants and isolated mesenchyme and epithelium; third, we specify the basics of obtaining still and live images; and finally, we bring these methods together by considering and briefly analyzing a lineage-labelling experiment.

Mechanism of MEN1 gene in radiation-induced pulmonary fibrosis in mice

ObjectiveTo investigate the regulatory mechanism of MEN1 gene in radiation-induced lung fibrosis in mice and provide a new theoretical basis for the clinical treatment of radiation pulmonary fibrosis. MethodsFirst, 80 C57BL/6 mice aged 8 weeks and weighing 18–22 g were selected, half of them were male and the other half were female. The mice were divided into control group and irradiation group (40 mice in each group) according to the method of the random number table. A radiation-induced lung fibrosis mouse model was established in which a single X-ray irradiation of 20 Gy was applied to the right lung in the irradiation group; H&E and Masson staining were used to verify whether the model was successful at 4, 8, 16 and 24 weeks after irradiation. The expression of MEN1, smooth muscle actin (α-SMA), Collagen-1 and transforming growth factor (TGF-β) in lung tissue were detected by Western blot and qPCR. Secondly, in the mouse embryonic fibroblast cell line (MEF) and mouse lung epithelial cell line (MLE-12), we constructed cell models of MEN1 knockout and interference separately with the irradiation of 10 Gy X-rays. The expression of α-SMA, Collagen-1, and TGF-β/Smads signaling pathway molecules was detected by qPCR. Finally, using the immunoprecipitation (IP) method, we can detect the interaction between Smad2 and the protein menin encoded by the MEN1 gene. ResultsThe results of the radiation pulmonary fibrosis model in mice showed that compared with the control group, the alveolar septum widens, the alveolar integrity decreases, the lung tissue slightly thickens, and a small amount of collagen deposits appear after 4–8 weeks in the model group. At twenty-fourth weeks, a large number of cells in the interstitial space of the lung tissue and a localized focal fibrosis area were observed. Further study found that radiation induced fibrogenic inflammatory cytokines TGF-β up-regulation, down-regulation of MEN1 gene expression, and then enhanced the expression of α-SMA and promotes the transformation of fibroblasts to myofibroblasts; At the same time, the expression of Collagen-1 was enhanced, which suggested that the extracellular matrix was overconcentrated and eventually promoted the formation of pulmonary fibrosis. In vitro, we found that knockout and interference of MEN1 gene can significantly enhance radiation-induced fibrosis, and up-regulate the expression of downstream molecules Smad2 and Smad3 of TGF-β signaling pathway, and down-regulate the expression of Smad7. Furthermore, it played an important role in regulating the process of radionuclide fibrosis. ConclusionMEN1 plays a key role in the formation of pulmonary fibrosis by regulating the secretion of TGF-β and the activation of TGF-β/Smads signaling pathway.

The mechanosensitive ion channel TRPV4 is a regulator of lung development and pulmonary vasculature stabilization.

Clinical observations and animal models suggest a critical role for the dynamic regulation of transmural pressure and peristaltic airway smooth muscle contractions for proper lung development. However, it is currently unclear how such mechanical signals are transduced into molecular and transcriptional changes at the cell level. To connect these physical findings to a mechanotransduction mechanism, we identified a known mechanosensor, TRPV4, as a component of this pathway. Embryonic mouse lung explants were cultured on membranes and in submersion culture to modulate explant transmural pressure. Time-lapse imaging was used to capture active changes in lung biology, and whole-mount images were used to visualize the organization of the epithelial, smooth muscle, and vascular compartments. TRPV4 activity was modulated by pharmacological agonism and inhibition. TRPV4 expression is present in the murine lung with strong localization to the epithelium and major pulmonary blood vessels. TRPV4 agonism and inhibition resulted in hyper- and hypoplastic airway branching, smooth muscle differentiation, and lung growth, respectively. Smooth muscle contractions also doubled in frequency with agonism and were reduced by 60% with inhibition demonstrating a functional role consistent with levels of smooth muscle differentiation. Activation of TRPV4 increased the vascular capillary density around the distal airways, and inhibition resulted in a near complete loss of the vasculature. These studies have identified TRPV4 as a potential mechanosensor involved in transducing mechanical forces on the airways to molecular and transcriptional events that regulate the morphogenesis of the three essential tissue compartments in the lung.

Cell type specific expression of Follistatin-like 1 (Fstl1) in mouse embryonic lung development.

Follistatin like-1 (Fstl1) is a secreted glycoprotein and can be up-regulated by TGF-β1. To better study the function of Fstl1 in lung development, we examined Fstl1 expression in the developing lung, in a cell type specific manner, using a tamoxifen inducible Fstl1-reporter mouse strain. Our results show that Fstl1 is ubiquitously expressed at saccular stage in the developing lung. At E18.5, Fstl1 expression is robust in most type of mesenchymal cells, including airway smooth muscle cells surrounding airways, vascular smooth muscle cells, endothelial cells, and vascular pericytes from blood vessel, but not PDGFRα+ fibroblasts in the distal alveolar sacs. Meanwhile, relative weak and sporadic signals of Fstl1 expression are observed in epithelium, including a subgroup of club cells in proximal airways and a few type II alveolar epithelial cells in distal airways. Our data help to understand the critical role of Fstl1 in lung development and lung disease pathogenesis.

Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter

BackgroundThe calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood.MethodsWe searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments.ResultsSeptin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7.ConclusionsOur results demonstrate septin 7 not only serving as a “cytoskeletal” protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.

Regulatory role of Shh signaling pathway in lung development in fetal mice

To investigate the regulatory role of classical Shh signaling pathway in the development of the epithelium and mesenchyme (bronchial cartilage and smooth muscles) during lung development in fetal mice. Immunohistochemical technique was used to detect the expression of Shh signaling pathway receptor Smo and Pdgfr-α in murine fetal lungs to explore the spatial and temporal characteristics of their expression. Based on the interstitial specificity of Pdgfr-α expression, we constructed a Pdgfr-α-cre to establish a E12.5 - E16.5 transgenic mice with specific knockout of the key Shh signaling molecule Smo in the pulmonary interstitium with tamoxifen induction. Immunofluorescence technique was used to observe the epithelium and mesenchyme (bronchial cartilage and smooth muscle) during fetal lung development in the transgenic mice to assess the role of Shh signaling pathway in the epithelial-to-mesenchymal (EMT) transition during the lung development. Smo was highly expressed in the epithelial and stromal lung tissues in the pseudoglandular stage and was gradually lowered over time with its distribution mainly in the interstitial tissues. Pdgfr-α was enriched in the distal lung epithelial and mesenchy tissues in early embryonic lungs and gradually migrated to the proximal stroma until becoming concentrated around the main bronchial proximal stroma. We successfully specific established mouse models of specific mesenchymal Smo knockout. Compared with the control group, the transgenic mice during E12.5-E16.5 showed significantly reduced lung the volume and bronchial branching with also decreased expression of the proximal epithelial P63 (P<0.05). The transgenic mice exhibited alterations in the expression of α-smooth muscle actin with delayed bronchial cartilage development and decreased expression of mucoprotein. The temporospatial specific expression of Shh signaling pathway plays an important role in developmental regulation of mouse embryonic lung epithelium and mesenchyme (bronchial cartilage and smooth muscle).

Lung Injury Repair by Transplantation of Adult Lung Cells Following Preconditioning of Recipient Mice

Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated that human and mouse embryonic lung tissue from the canalicular stage of development are enriched with lung progenitors, and that a single cell suspension of canalicular lungs can be used for transplantation, provided that lung progenitor niches in the recipient mice are vacated by strategies similar to those used in bone marrow transplantation. Considering the ethical limitations associated with the use of fetal cells, we investigated here whether adult lungs could offer an alternative source of lung progenitors for transplantation. We show that intravenous infusion of a single cell suspension of adult mouse lungs from GFP+ donors, following conditioning of recipient mice with naphthalene and subsequent sublethal irradiation, led to marked colonization of the recipient lungs, at 6–8 weeks post‐transplant, with donor derived structures including epithelial, endothelial, and mesenchymal cells. Epithelial cells within these donor‐derived colonies expressed markers of functionally distinct lung cell types, and lung function, which is significantly compromised in mice treated with naphthalene and radiation, was found to be corrected following transplantation. Dose response analysis suggests that the frequency of patch forming cells in adult lungs was about threefold lower compared to that found in E16 fetal lungs. However, as adult lungs are much larger, the total number of patch forming cells that can be collected from this source is significantly greater. Our study provides proof of concept for lung regeneration by adult lung cells after preconditioning to vacate the pulmonary niche. stem cells translational medicine 2018;7:68–77

3D culture of multipotent Sox9+ mouse embryonic lung progenitors: Isolation, Expansion and Cryopreservation

3D culture of multipotent Sox9+ mouse embryonic lung progenitors: Isolation, Expansion and Cryopreservation

Claudin18 associated with corticosteroid-induced expression of surfactant proteins in pulmonary epithelial cells

Objective: The present study was undertaken to test the hypothesis that if Cldn18 is associated with lung maturation and play a key role in the corticosteroid-induced expression of surfactant proteins.Methods: Mouse embryonic lung tissue at different developmental stages (E14.5, E16.5 and E18.5) was collected to detect the temporal-spatial expression of Cldn18 by Western blot and immunofluorescence staining. Dexamethasone + cAMP + isobutylmethylxanthine (DCI), a commonly used agent for inducing maturation of alveolar epithelial cell in vitro, was used to mimic the roles of corticosteroid. Mouse alveolar epithelial cell line MLE12 treated with or without DCI were harvested, expression of Cldn18 and surfactant proteins were assessed by real-time RT-PCR. For Si-RNA experiments, MLE-12 was transfected with control siRNA or Cldn18 siRNA for 24 h before DCI treatment. Expression of Cldn18 and surfactant proteins was assessed by real-time RT-PCR.Results: Expression of Cldn18 was detected in peripheral lung epithelial cells, and increased expression was detected from E14.5 to E18.5 during lung development. Expression of Cldn18 and surfactants (SPA, SPB and SPC) were significantly induced in MLE12 after DCI treatment. Silencing of Cldn18 effectively suppressed the DCI-induced expression of SPB and SPC but not SPA.Conclusion: Our results demonstrated that expression of Cldn18 is associated with peripheral lung epithelial cell maturation and play a key role in the corticosteroid-induced expression of surfactant proteins.

Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids.

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.

In vivo imaging of the Muc5b gel-forming mucin

Gel-forming mucins are macromolecules produced by goblet cells and responsible for the mucus gel formation. Changes in goblet cell density and in gel-forming mucin production have emerged as sensitive indicators for mucosal diseases. A Muc5b-GFP tagged reporter mouse was used to assess Muc5b production in mouse tissues by immunofluorescence microscopy and fluorescent activity using stereromicroscopy and probe-based confocal laser endomicroscopy. Muc5b production was followed longitudinally by recording the fluorescent activity in vagina and in embryonic lung explants under stimulation by interleukin 13. We show that the GFP is easily visualized in the mouse adult ear, nose, trachea, gallbladder, and cervix. Live Muc5b is also easily monitored in the nasal cavity, trachea and vagina where its production varies during the estrus cycle with a peak at the proestrus phase and in pregnant mice. Explant culture of reporter mouse embryonic whole lung shows that interleukin 13 stimulates Muc5b production. The transgenic Muc5b-GFP mouse is unique and suitable to study the mechanisms that regulate Muc5b production/secretion and mucous cell differentiation by live imaging and can be applied to test drug efficacy in mucosal disease models.

Development of a genetically modifiable epithelial in-vitro culture system from human embryonic lung epithelial stem cells: towards human lung regeneration in end-stage respiratory failure

BackgroundRegeneration of healthy lung tissue in patients with end-stage respiratory disease would cure disease. Therefore, a detailed understanding of lung development is needed. The most important epithelial stem-cell population in developing lungs is found in distal branching tips, and these Sox9+ lung epithelial stem cells (LESCs) generate all epithelial lineages. Our objective was to develop a self-renewing, genetically modifiable epithelial in-vitro culture system from human embryonic LESCs. MethodsHuman embryonic LESCs were characterised using genome-wide transcriptional analysis (RNA-seq, Illumina, San Diego, CA, USA) and immunohistochemistry at 5–20 weeks postconception. LESCs were microdissected, and self-renewing expansion in three-dimensional (3D) organoid culture was established empirically. We assessed similarity between cultured and fresh LESCs via RNA-seq and immunohistochemistry. Organoids were differentiated in vitro, or in vivo by xenotransplantation into bleomycin-injured mouse lungs or kidney capsule. SOX9 was deleted with CRISPR–Cas9. FindingsRNA-seq of LESCs identified broad-scale transcriptional differences between mouse and human embryonic lung stem cells. Human LESCs were successfully expanded for more than 10 months as karyotypically stable 3D organoids using a combination of seven signalling molecules. The LESC stem-cell markers, transcriptome, and organoid morphology were maintained throughout the culture period. Bronchiolar and alveolar differentiation was achieved in vitro and in vivo. Moreover, xenotransplantation of organoids into bleomycin-injured adult mouse lungs was extremely efficient. Knocking out SOX9 led to a loss-of-self-renewing phenotype. InterpretationOur novel genetically modifiable human embryonic lung culture system enables for the first time, to our knowledge, the in-vitro study of human lung development and disease. We anticipate that this system will transform lung regenerative medicine by guiding development of improved protocols for induced pluripotent stem-cell differentiation and adult stem-cell manipulation in vivo, benefiting patients with end-stage respiratory disease. Furthermore, our lung injury–xenotransplantation protocol provides the first method for efficient future cell therapy. FundingWellcome Trust (PhD programme for clinicians), Medical Research Council, Addenbrooke's Charitable Trust.

SLICE: determining cell differentiation and lineage based on single cell entropy.

A complex organ contains a variety of cell types, each with its own distinct lineage and function. Understanding the lineage and differentiation state of each cell is fundamentally important for the ultimate delineation of organ formation and function. We developed SLICE, a novel algorithm that utilizes single-cell RNA-seq (scRNA-seq) to quantitatively measure cellular differentiation states based on single cell entropy and predict cell differentiation lineages via the construction of entropy directed cell trajectories. We validated our approach using three independent data sets with known lineage and developmental time information from both Homo sapiens and Mus musculus. SLICE successfully measured the differentiation states of single cells and reconstructed cell differentiation trajectories that have been previously experimentally validated. We then applied SLICE to scRNA-seq of embryonic mouse lung at E16.5 to identify lung mesenchymal cell lineage relationships that currently remain poorly defined. A two-branched differentiation pathway of five fibroblastic subtypes was predicted using SLICE. The present study demonstrated the general applicability and high predictive accuracy of SLICE in determining cellular differentiation states and reconstructing cell differentiation lineages in scRNA-seq analysis.