Isolation and culture of mouse alveolar type II cells to study type II to type I cell differentiation.
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ABSTRACT:
SUBMITTER: Chen Q
PROVIDER: S-EPMC7788236 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells.
| S-EPMC4393197 | biostudies-other
Project description:Resident stem/progenitor cells in the lung are important for tissue homeostasis and repair. However, a progenitor population for alveolar type II (ATII) cells in adult human lungs has not been identified. The aim of this study is to isolate progenitor cells from adult human lungs with the ability to differentiate into ATII cells. We isolated colony-forming cells that had the capability for self-renewal and the potential to generate ATII cells in vitro. These undifferentiated progenitor cells expressed surface markers of mesenchymal stem cells (MSCs) and surfactant proteins associated with ATII cells, such as CD90 and pro-surfactant protein-C (pro-SP-C), respectively. Microarray analyses indicated that transcripts associated with lung development were enriched in the pro-SP-C(+)/CD90(+) cells compared with bone marrow-MSCs. Furthermore, pathological evaluation indicated that pro-SP-C and CD90 double-positive cells were present within alveolar walls in normal lungs, and significantly increased in ATII cell hyperplasias contributing to alveolar epithelial repair in damaged lungs. Our findings demonstrated that adult human lungs contain a progenitor population for ATII cells. This study is a first step toward better understanding of stem cell biology in adult human lung alveoli.
| S-EPMC3125101 | biostudies-literature
Project description:Regenerative medicine has continued to progress for lung biology and lung diseases. Efforts have focused on a variety of different applications for pluripotent stem cells. Several groups have reported successful methods for inducing differentiation of induced pluripotent stem cells (iPSCs) into the airway epithelium such as alveolar epithelium type II (ATII). However, differentiation efficiency varies among reports and improvements are needed. In the present paper, we propose a novel method for elimination of residual undifferentiated murine iPSCs using JQ1, a potent inhibitor of bromodomain (BRD) and extraterminal domain (BET) family proteins, for efficient differentiation into ATII. First, the murine iPSC line 20D-17 was induced to differentiate into ATII over a period of 26 days (days 0-26) using previously reported embryoid body seeding and stepwise differentiation methods. mRNA expressions of differentiation markers including surfactant protein C (Sftpc) were confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) results, and 17% of the cells were shown positive for prosurfactant protein C (proSPC) in flow cytometry analysis. Next, those cells were cultured three-dimensionally in Matrigel for an additional 14 days (days 26-40), during which JQ1 was added for 4 days (days 28-32) to remove residual undifferentiated iPSCs. As a result, on day 40, the mRNA expression level of Sftpc in the three-dimensional culture was maintained at the same level as on day 26 and shown to be further increased by the addition of JQ1, with 39% of the cells found to express proSPC, showing that differentiation efficiency could be further increased. Three-dimensional culture with BRD4 inhibition by JQ1 improved the differentiation induction efficiency to ATII by removing residual undifferentiated murine iPSCs during the differentiation induction process.
| S-EPMC6948319 | biostudies-literature
Project description:In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids-specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment.
| S-EPMC2599825 | biostudies-other
Project description:Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 'alveolar' cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham's F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.
| S-EPMC5085087 | biostudies-literature
Project description:Human surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. SP-A is synthesized and secreted by alveolar type II (ATII) cells, one of the two cell types of the distal lung epithelium (ATII and ATI). We have shown that miRNA interactions with sequence polymorphisms on the SP-A mRNA 3'UTRs mediate differential expression of SP-A1 and SP-A2 gene variants in vitro. In the present study, we describe a physiologically relevant model to study miRNA regulation of SP-A in human ATII. For these studies, we purified and cultured human ATII on an air-liquid interface matrix (A/L) or plastic wells without matrix (P). Gene expression analyses confirmed that cells cultured in A/L maintained the ATII phenotype for over 5 days, whereas P-cultured cells differentiated to ATI. When we transfected ATII with siRNAs to inhibit the expression of Drosha, a critical effector of miRNA maturation, the levels of SP-A mRNA and protein increased in a time dependent manner. We next characterized cultured ATII and ATI by studying expression of 1,066 human miRNAs using miRNA PCR arrays. We detected expression of >300 miRNAs with 24 miRNAs differentially expressed in ATII versus ATI, 12 of which predicted to bind SP-A 3'UTRs, indicating that these may be implicated in SP-A downregulation in ATI. Thus, miRNAs not only affect SP-A expression, but also may contribute to the maintenance of the ATII cell phenotype and/or the trans-differentiation of ATII to ATI cells, and may represent new molecular markers that distinguish ATII and ATI.
| S-EPMC4197128 | biostudies-literature
Project description:We have examined phospholipid-transfer activities in cytosols from rat and mouse whole lung, isolated rat alveolar type II cells and alveolar type II cell-derived mouse pulmonary adenomas. We report an enrichment in phosphatidylcholine and phosphatidylglycerol (but not phosphatidylinositol) protein-catalysed transfer in the type II cell and adenoma cytosols compared with the whole-lung cytosols. The activities from these cytosols were resolved using column chromatofocusing, which clearly demonstrated the presence of a phosphatidylcholine-specific transfer protein in each of the four tissues. In addition, two proteins (rat) or three proteins (mouse) catalysing both phosphatidylcholine and phosphatidylglycerol transfer were resolved from whole lung, whereas in both the rat isolated alveolar type II cells and the mouse type II cell-derived adenomas one of these less specific proteins is not present.
| S-EPMC1152446 | biostudies-other
Project description:Inhaled nanoparticles have a high deposition rate in the alveolar units of the deep lung. The alveolar epithelium is composed of type-I and type-II epithelial cells (ATI and ATII respectively) and is bathed in pulmonary surfactant. The effect of native human ATII cell secretions on nanoparticle toxicity is not known. We investigated the cellular uptake and toxicity of silver nanowires (AgNWs; 70 nm diameter, 1.5 ?m length) with human ATI-like cells (TT1), in the absence or presence of Curosurf® (a natural porcine pulmonary surfactant with a low amount of protein) or harvested primary human ATII cell secretions (HAS; containing both the complete lipid as well as the full protein complement of human pulmonary surfactant i.e. SP-A, SP-B, SP-C and SP-D). We hypothesised that Curosurf® or HAS would confer improved protection for TT1 cells, limiting the toxicity of AgNWs. In agreement with our hypothesis, HAS reduced the inflammatory and reactive oxygen species (ROS)-generating potential of AgNWs with exposed TT1 cells. For example, IL-8 release and ROS generation was reduced by 38% and 29%, respectively, resulting in similar levels to that of the non-treated controls. However in contrast to our hypothesis, Curosurf® had no effect. We found a significant reduction in AgNW uptake by TT1 cells in the presence of HAS but not Curosurf. Furthermore, we show that the SP-A and SP-D are likely to be involved in this process as they were found to be specifically bound to the AgNWs. While ATI cells appear to be protected by HAS, evidence suggested that ATII cells, despite no uptake, were vulnerable to AgNW exposure (indicated by increased IL-8 release and ROS generation and decreased intracellular SP-A levels one day post-exposure). This study provides unique findings that may be important for the study of lung epithelial-endothelial translocation of nanoparticles in general and associated toxicity within the alveolar unit.
| S-EPMC4765325 | biostudies-literature
Project description:The alveolar epithelium is composed of type I cells covering most of the gas-blood exchange surface and type II cells secreting surfactant that lowers surface tension of alveoli to prevent alveolar collapse. Here, we have identified a subgroup of type II cells expressing a higher level of cell surface molecule CD44 (CD44high type II cells) that composed ~3% of total type II cells in 5-10-wk-old mice. These cells were preferentially apposed to lung capillaries. They displayed a higher proliferation rate and augmented differentiation capacity into type I cells and the ability to form alveolar organoids compared with CD44low type II cells. Moreover, in aged mice, 18-24 mo old, the percentage of CD44high type II cells among all type II cells was increased, but these cells showed decreased progenitor properties. Thus CD44high type II cells likely represent a type II cell subpopulation important for constitutive regulation of alveolar homeostasis.
| S-EPMC5538873 | biostudies-other
Project description: Not available
| S-EPMC7287157 | biostudies-literature