Background: Myofibroblasts play a crucial role in tissue repair. The functional similarities and differences between myofibroblasts and fibroblasts are not fully understood because they have not been separately isolated from a living body. The purpose of this study was to establish a method for the direct isolation of myofibroblasts and fibroblasts from injured lungs by using fluorescence-activated cell sorting and to compare their functions. Results: We demonstrated that lineage-specific cell surface markers (lin), such as CD31, CD45, CD146, EpCAM (CD326), TER119, and Lyve-1 were not expressed in myofibroblasts or fibroblasts. Fibroblasts of bleomycin-injured lungs and saline-treated lungs were shown to be enriched in lin^neg Sca-1^high, and myofibroblasts of bleomycin-injured lungs were shown to be enriched in lin^neg Sca-1^low CD49e^high. Results from in-vitro proliferation assays indicated in-vitro proliferation of fibroblasts but not myofibroblasts of bleomycin-injured lungs and of fibroblasts of saline-treated lungs. However, fibroblasts and myofibroblasts might have a low proliferative capacity in vivo. Analysis of genes for collagen and collagen synthesis enzymes by qRT-PCR showed that the expression levels of about half of the genes were significantly higher in fibroblasts and myofibroblasts of bleomycin-injured lungs than in fibroblasts of saline-treated lungs. By contrast, the expression levels of 8 of 11 chemokine genes of myofibroblasts were significantly lower than those of fibroblasts. Conclusions: This is the first study showing a direct isolation method of myofibroblasts and fibroblasts from injured lungs. We demonstrated functional similarities and differences between myofibroblasts and fibroblasts in terms of both their proliferative capacity and the expression levels of genes for collagen, collagen synthesis enzymes, and chemokines. Thus, this direct isolation method has great potential for obtaining useful information from myofibroblasts and fibroblasts.

Histological examination using the light microscopy is currently the gold standard for life science research and diagnostics. However, magnified observations are limited because of the limitations intrinsic to light microscopy. Thus, a dual approach, known as correlative light and electron microscopy (CLEM), has emerged, although several technical challenges remain in terms of observing myriad stored paraffin sections. Previously, we developed the NanoSuit method, which enabled us to keep multicellular organisms alive/wet in the high vacuum of a scanning electron microscope by encasing the sample in a thin, vacuum-proof membrane. The approach uses the native extracellular substance (ECS) or an ECS-mimicking substance to polymerize a membrane by plasma or electron beam irradiation. Since the resulting NanoSuit is flexible and dense enough to prevent a living organism’s bodily gas and liquids from evaporating (which we refer to as the “surface shield enhancer” (SSE) effect), it works like a miniature spacesuit with sufficient electron conductivity for an SEM observation. Here, we apply the NanoSuit method to CLEM analysis of paraffin sections. Accordingly, the NanoSuit method permits the study of paraffin sections using CLEM at low and high magnification, with the following features: (i) the integrity of the glass slide is maintained, (ii) three-dimensional microstructures of tissue and pathogens are visualized, (iii) nuclei and 3,3′-diaminobenzidine-stained areas are distinct because of gold chloride usage, (iv) immunohistochemical staining is quantitative, and (v) contained elements can be analyzed. Removal of the SSE solution after observation is a further advantage, as this allows slides to be restained and stored. Thus, the NanoSuit method represents a novel approach that will advance the field of histology.

Background: Lung fibrosis is a serious life-threatening condition whose manifestation varies according to the localization and characteristics of fibroblasts, which are considered heterogeneous. Therefore, to better understand the pathology and improve diagnosis and treatment of this disease, it is necessary to elucidate the nature of this heterogeneity and identify markers for the accurate classification of human lung fibroblast subtypes. Methods: We characterized distinct mouse lung fibroblast subpopulations isolated by fluorescence-activated cell sorting (FACS) and performed microarray analysis to identify molecular markers that could be useful for human lung fibroblast classification. Based on the expression of these markers, we evaluated the fibroblast-like cell subtype localization in normal human lung samples and lung samples from patients with idiopathic pulmonary fibrosis (IPF). Results: Mouse lung fibroblasts were classified into Sca-1^high fibroblasts and Sca-1^low fibroblasts by in vitro biological analyses. Through microarray analysis, we demonstrated CD248 and integrin alpha-8 (ITGA8) as cell surface markers for Sca-1^high fibroblasts and Sca-1^low fibroblasts, respectively. In mouse lungs, Sca-1^high fibroblasts and Sca-1^low fibroblasts were localized in the collagen fiber-rich connective tissue and elastic fiber-rich connective tissue, respectively. In normal human lungs and IPF lungs, two corresponding major fibroblast-like cell subtypes were identified: CD248^highITGA8^low fibroblast-like cells and CD248^lowITGA8^high fibroblast-like cells, localized in the collagen fiber-rich connective tissue and in the elastic fiber-rich connective tissue, respectively. Conclusion: CD248^highITGA8^low fibroblast-like cells and CD248^lowITGA8^high fibroblast-like cells were localized in an almost exclusive manner in human lung specimens. This human lung fibroblast classification using two cell surface markers may be helpful for further detailed investigations of the functions of lung fibroblast subtypes, which can provide new insights into lung development and the pathological processes underlying fibrotic lung diseases.

Enhancer of Zeste homologue 2 (EZH2) overexpression is associated with tumor proliferation, metastasis, and poor prognosis. Targeting and inhibition of EZH2 is a potentially effective therapeutic strategy for head and neck squamous cell carcinoma (HNSCC). We analyzed EZH2 mRNA expression in a well-characterized dataset of 230 (110 original and 120 validation cohorts) human head and neck cancer samples. This study aimed to investigate the effects of inhibiting EZH2, either via RNA interference or via pharmacotherapy, on HNSCC growth. EZH2 upregulation was significantly correlated with recurrence (p < 0.001) and the methylation index of tumor suppressor genes (p < 0.05). DNMT3A was significantly upregulated upon EZH2 upregulation (p = 0.043). Univariate analysis revealed that EZH2 upregulation was associated with poor disease-free survival (log-rank test, p < 0.001). In multivariate analysis, EZH2 upregulation was evaluated as a significant independent prognostic factor of disease-free survival (hazard ratio: 2.085, 95% confidence interval: 1.390–3.127; p < 0.001). Cells treated with RNA interference and DZNep, an EZH2 inhibitor, showed the most dramatic changes in expression, accompanied with a reduction in the growth and survival of FaDu cells. These findings suggest that EZH2 upregulation is correlated with tumor aggressiveness and adverse patient outcomes in HNSCC. Evaluation of EZH2 expression might help predict the prognosis of HNSCC patients.

The potential of neural stem and progenitor cell (NSPC) transplantation in neurodegenerative disease raises a concern about immunosuppressive agents and opportunistic neurotropic pathogens that may interfere with engraftment. Cytomegalovirus (CMV) is an important opportunistic pathogen infecting the central nervous system, where it may remain latent for life, following transplacental transmission. Cyclosporine (Cs), an immunosuppressive drug used in organ transplantation, where its use is associated with CMV reactivation, suppressed murine CMV (MCMV) infection in cultured NSPCs but not in fibroblasts. This activity of Cs appears to be mediated via cyclophilin (CyP) rather than via calcineurin. First, the calcineurin-specific inhibitor FK506 failed to suppress replication. Second, the CyP-specific inhibitor NIM811 strongly suppressed replication in NSPC. NSPCs maintained in the presence of NIM811 retained viral genomes for several weeks without detectable viral gene expression or obvious deleterious effects. The withdrawal of NIM811 reactivated viral replication, suggesting that the inhibitory mechanism was reversible. Finally, inhibition of endogenous CyP A (CyPA) by small interfering RNA also inhibited replication in NSPCs. These results show that MCMV replication depends upon cellular CyPA pathways in NSPCs (in a specific cell type-dependent fashion), that CyPA plays an important role in viral infection in this cell type, and that inhibition of viral replication via CyP leads to persistence of the viral genome without cell damage. Further, the calcineurin-signaling pathway conferring immunosuppression in T cells does not influence viral replication in a detectable fashion.