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Single cell transcriptomic profiling of pluripotent stem cell-derived alveolar type 2 cells expressing SFTPC-tdTomato cultured in 3D spheres, 2D submerged culture, and air-liquid interface culture with and without cigarette smoke exposure

ABSTRACT: We performed transcriptomic profiling of cells derived from human induced pluripotent stem cells (iPSCs) using previously described distal lung directed differentiation protocol to generate alveolar type 2 cells (iAT2s). We used the SPC2-ST-B2 (“SPC2”) line containing a SFTPC-tdTomato knock in reporter. iAT2s were cultured as 3D spheres (3D), on Transwells in 2D submerged conditions (2D), or at ALI for a total of 10 days post passaging. iAT2s in ALI culture were exposed to cigarette smoke 12 hours prior to cell collection (Smoke) or air (ALI). On day 10, and at 12 hours post cigarette smoke exposure, cultures were dissociated to single cells and live cells were sorted on calcein blue on a Mo-Flo Astrios Cell Sorter. Capture and library preparation was performed using a 10X Chromium system. Libraries were sequenced using a NextSeq 500. We find that ALI culture promotes maturation of iAT2s compared to 3D, and that iAT2s mount an inflammatory and xenobiotic metabolism response to cigarette smoke exposure at ALI.

ORGANISM(S): Homo sapiens

PROVIDER: GSE184142 | GEO | 2022/04/30

REPOSITORIES: GEO

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Bulk transcriptomic profiling of pluripotent stem cell-derived alveolar type 2 cells (iAT2s) cultured in spheres and at air-liquid interface and human HTII-280-sorted AT2s from a pediatric and adult donor lungs cultured in spheres

Project description:We performed bulk transcriptomic profiling of pluripotent stem cell-derived alveolar type 2 cells (iAT2s) and HTII-280-sorted human AT2s cultured with CK+DCI media. We used previously described distal lung directed differentiation protocol to generate alveolar type 2 cells (iAT2s). We used the SPC2-ST-B2 (“SPC2”) line containing a SFTPC-tdTomato knock in reporter. iAT2s were cultured in 3D spheres and at air-liquid interface. Human AT2s were HTII-280-sorted from a pediatric donor lung (13 months old) and an adult, nonsmoker human lung (32 years old) and cultured as spheres in the presence of MRC5s. After deconvolution, we find that iAT2s cultured in ALI correlate more closely to primary cultured AT2s than do iAT2s cultured in 3D spheres on the basis of lung epithelial gene expression.

2022-04-30 | GSE184143 | GEO

Direct exposure to SARS-CoV-2 and cigarette smoke increases infection severity and alters the stem cell-derived airway repair response

Project description:To study the effect of cigarette smoke exposure on Sars-Cov2 infection, we directly exposed mucociliary air-liquid interface (ALI) cultures derived from primary human nonsmoker airway basal stem cells (ABSCs) to short term cigarette smoke and infected them with live SARS-CoV-2. We set out to examine the underlying mechanisms governing the increased susceptibility of cigarette smoke exposed ALI cultures to SARS-CoV-2 infection by usingle cell profiling of the cultures, which showed that interferon response genes were induced in SARS-CoV-2 infected airway epithelial cells in ALI cultures but smoking exposure together with SARS-CoV-2 infection reduced the interferon response.

2020-11-17 | GSE161089 | GEO

Comprisons of lung tissue gene expression from AKR and C57BL/6 mice exposed to Cigarette smoke and LPS in ARDS/ALI mice model

Project description:Our previous study showed that AKR and C57BL/6 mice to cigarette smoke increased lipopolysaccharide (LPS)- induced increased lung vascular permeability. Viremic AKR mice were more susceptible to LPS-induced ALI than C57 on prolonged exposure. In this study we compared the global gene expression patterns to determine the genetic basis for the strain dependent responses to cigarette smoke and LPS-induced ALI. We found AKR transcriptomic responses didnot overlap with C57 mice and AKR mice showed increased responses compared to C57 mice. Global gene expression analysis using microarray was used to dermine the mechanisms of strain dependent responses to cigarette smoking and LPS mediated ALI

2023-05-28 | GSE193958 | GEO

Bulk RNA-seq of CRISPRi ADGRG6-kd iAT2 following cigarette smoke exposure

Project description:We performed bulk transcriptomic profiling of induced human pluripotent stem cells (iPSCs)-derived type 2 alveolar epithelial cells (iAT2). iPSCs stably expressed CRISPRi (dCas9-KRAB) under the control of doxycyline. iAT2s were transduced with a lentivirus expressing gRNA targeting the transcriptional start site of ADGRG6. Cells were treated with or without doxycyline to intiate CRISPRi-knockdown. Cells were plated at an air-liquid interface, then subsequently exposed to air or 5% cigarette smoke using a VitroCell smoke robot. Cells were harvested for bulk RNA sequencing 8 hours post cigarette smoke exposure

2023-09-20 | GSE223077 | GEO

Global transcriptomic analyses of pluripotent stem cell-derived type II alveolar epithelial-like cells at 1 and 4 days after infection with SARS-CoV-2

Project description:We performed transcriptomic profiling of cells derived from human pluripotent stem cells (PSCs) using our previously described distal lung directed differentiation protocol to generate alveolar type II epithelial-like cells (iAT2s). We used the SPC2 human iPSC line containing a SFTPC-tdTomato knock-in reporter. SFTPC-tdTomato+ iAT2s were sorted on day 41 and again on day 69 of differentiation. iAT2s were single-cell passaged in self-renewing 3D alveolosphere culture approximately every 2 weeks through day 194 of differentiation. On day 208, iAT2s were single-cell passaged onto Transwell inserts. Apical media was removed on day 210 to initiate air-liquid interface culture. On day 218, 6 replicate wells of iAT2s were exposed to SARS-CoV-2 in an apical inoculum and 3 replicate wells were exposed to mock. On day 219, three mock and three post-infection samples (1 dpi) were collected. Three additional post-infection samples (4 dpi) were collected on day 222. We find that SARS-CoV-2 infected iAT2s express a rapid global transcriptomic change characterized by a shift to an inflammatory phenotype, time-dependent epithelial interferon responses, and rapid loss of the mature lung alveolar epithelial program.

2020-09-21 | GSE153277 | GEO

Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture

Project description:Cigarette smoke (CS) is the leading cause to develop COPD. Therefore, we investigated the pathologic effects of whole CS on the differentiation of primary small airway epithelial cells (SAEC), from three healthy donors and three COPD patients, cultured under ALI (air-liquid interface) conditions. The analysis of the epithelial physiology demonstrated that CS impaired barrier formation and reduced cilia beat activity. Although, COPD-derived ALI cultures preserved some features known from COPD patients, the CS-induced effects were similarly pronounced in ALI cultures from patients compared to healthy controls. A RNA sequencing analysis revealed the deregulation for marker genes for basal cells and secretory cells upon CS exposure. The comparison between gene signatures obtained from our in vitro model (CS vs. air) with a published data set from human epithelial brushes (smoker vs. non-smoker) reveals a high degree of similarity between the deregulated genes and pathways. ALI culture has been generated utilising cells obtained from 3 COPD and 3 healthy subjects. Subsequently cultures have been treated with CS and air respectively for 33 days. Gene expression profiles were generated using next generation sequencing.

2019-12-31 | GSE135188 | GEO

Repeated exposure and switching study of cigarette smoke and next generation tobacco product using 3D human bronchial epithelial cells

Project description:Next generation product such as heat-not-burn type product and e-cigarette emit lower yields of chemical constituents than cigarette because combustion of tobacco leaves is not associated with their vapor generation. Therefore it is expected that the usage of these product bring beneficial impact on public health. However, risk-reduced potential as well as risk concerns in long-term use of these products has not yet been fully understood. In this study, we intended to estimate the risk reduced potential of our proprietary novel tobacco vapor product (NTV) by repeated exposure of in vitro 3D bronchial epithelial cells to NTV vapor in comparison with repeated exposure to cigarette smoke. In addition, to reflect the realistic situation of smokers, exposure switching from cigarette smoke to NTV vapor and cigarette smoke exposure cessation were conducted.

2020-03-31 | E-MTAB-8155 | biostudies-arrayexpress

Single-cell analysis of Kaposi’s sarcoma-associated herpesvirus infection in three-dimensional air-liquid interface culture model

Project description:The oral cavity has previously been identified as the major site for transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV), but how KSHV establishes infection and replication in the oral epithelia remains unclear. Here, we report a KSHV spontaneous lytic replication model using fully differentiated, three-dimensional (3D) oral epithelial organoids at an air-liquid interface (ALI). This model revealed that KSHV infected the oral epithelia when the basal epithelial cells were exposed by damage. Unlike two-dimensional (2D) cell culture, 3D oral epithelial organoid ALI culture allowed high levels of spontaneous KSHV lytic replication, where lytically replicating cells were enriched at the superficial layer of epithelial organoid. Single cell RNA sequencing (scRNAseq) showed that KSHV infection induced drastic changes of host gene expression in infected as well as uninfected cells at the different epithelial layers, resulting in altered epithelial differentiation and morphogenesis. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host and viral gene expression compared to latency or lytic replication. This study demonstrates an in vitro 3D epithelial organoid ALI culture model that recapitulates KSHV infection in the oral cavity, where KSHV undergoes the epithelial differentiation-dependent spontaneous lytic replication with a unique cell population carrying distinct viral gene expression.

2022-07-28 | GSE184257 | GEO

Cystathionine beta synthase (CBS) Over expression vs PLenti6 Expression Vector Human Bronchial Epithelial Cells (HBEC)

Project description:Cystathionine Beta Synthase (CBS), an enzyme that utilizes a methionine precursor to generate glutathione (GSSH), has been implicated in the mitigation of ROS production in COPD lung. Additionally, post-translational modification analysis on mouse and human cells treated with cigarette smoke showed the unique presence of mono- and di-methylated histone H3 lysine 27 that was not seen in controls, suggesting that cigarette smoke could lead to loss of Polycomb Repressive Complex 2 (PRC2) activity. To elucidate CBS as a potential upstream target for PRC2 inhibition, we overexpressed CBS in Human Bronchial Epithelial Cells (HBECs), grew them as 2D cultures, then conducted bulk RNA seq analysis.

2022-06-30 | GSE183858 | GEO

A comparison of mono-culture and co-culture model containing fibroblasts

Project description:Organotypic three dimensional cultures of epithelial cells are grown at the air–liquid interface (ALI) and resemble the in vivo counterparts. Although the complexity of in vivo cellular responses could be better manifested in co-culture models in which additional cell-types such as fibroblasts were incorporated, the presence of another cell-type could mask the response of the other. This study reports the impact of whole combustible cigarette smoke (CS) on organotypic mono- and co-culture models to evaluate the relevancy of organotypic models for toxicological assessment of aerosols. Two organotypic bronchial models were directly exposed to low and high concentrations of CS of the reference research cigarette 3R4F: mono-culture of bronchial epithelial cells without fibroblasts (BR) and co-culture with fibroblasts (BRF) models. Adenylate kinase-based cytotoxicity, cytochrome P450 (CYP) 1A1/1B1 activity, tissue histology, and concentrations of secreted mediators in the basolateral media, as well as transcriptomes were evaluated following the CS exposure. The results demonstrated similar impact of CS on the AK-based cytotoxicity, CYP1A1/1B1 activity, and tissue histology in both models. However, a greater number of secreted mediators were found in the basolateral media of the mono-culture than in the co-culture models. Furthermore, annotation analysis and network-based systems biology analysis of the transcriptomic profiles indicated a more prominent cellular stress and tissue damage following CS in the mono-culture epithelium model without fibroblasts. Finally, our results indicated that the in vivo smoking-induced xenobiotic metabolism response in the bronchial epithelial cells was better reflected on the in vitro co-culture model upon CS exposure.

2015-06-12 | E-MTAB-3462 | biostudies-arrayexpress

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