Human Pluripotent Stem Cell-Derived Alveolar Epithelial Cells as a Tool to Assess Cytotoxicity of Particulate Matter and Cigarette Smoke Extract

Abstract

Human pluripotent stem cells (hPSCs) can give rise to a vast array of differentiated derivatives, which have gained great attention in the field of in vitro toxicity evaluation. We have previously demonstrated that hPSC-derived alveolar epithelial cells (AECs) are phenotypically and functionally similar to primary AECs and could be more biologically relevant alternatives for assessing the potential toxic materials including in fine dust and cigarette smoking. Therefore, in this study, we employed hPSC-AECs to evaluate their responses to exposure of various concentrations of diesel particulate matter (dPM), cigarette smoke extract (CSE) and nicotine for 48 hrs in terms of cell death, inflammation, and oxidative stress. We found that all of these toxic materials significantly upregulated the transcription of pro-inflammatory cytokines such as IL-1α, IL-β, IL-6, and TNF-α. Furthermore, the exposure of dPM (100 μg/mL) strongly induced upregulation of genes related with cell death, inflammation, and oxidative stress compared with other concentrations of CSE and nicotine. These results suggest that hPSC-AECs could be a robust in vitro platform to evaluate pulmotoxicity of various air pollutants and harmful chemicals.

Keywords: Alveolar epithelial cells (AECs); Cigarette smoking extract (CSE); Cytotoxicity; Diesel particulate matter (dPM); Human pluripotent stem cells (hPSCs).

Fig. 1.. Generation of hPSC-AECs.

(A) Schematic diagram of stepwise AEC differentiation from hPSCs and representative images of AEC development. Scale bars: 100 μm. (B, C) Representative FACS plots based on expression of NKX2.1, CPM, EPCAM, STFPB, STFPC, T1α and AQP5 in day 14 hPSC-AEPs (B) and 25 (C) hPSC-AECs, respectively. hPSC, human pluripotent stem cell; AEC, alveolar epithelial cells; AEP, alveolar epithelial progenitors; EPCAM, epithelial cell adhesion molecule.

Fig. 2.. Effect of dPM, Nicotine and CSE on cell viability.

hpAECs were treated with various concentrations of dPM (50, 100, and 200 μg/mL), Nicotine (1, 10, and 100 μM) and CSE (1%, 2%, and 3%) and incubated for 48 hrs. Cell viability was measured by the MTS assay. Data expressed a mean±SD. * p<0.05, ** p<0.01 vs Control. dPM, diesel particulate matter; CSE, cigarette smoke extract; AEC, alveolar epithelial cells.

Fig. 3.. Effects of dPM exposure on cell death, inflammation and oxidative stress in hPSC-AECs.

hPSC-AECs were treated with the indicated concentrations of dPM for 48 hrs. Transcript levels of cell death (A), inflammation (B) and oxidative stress (C)-related genes were measured by quantitative real-time PCR. Data expressed a mean±SD. * p<0.05, ** p<0.01 vs Control. dPM, diesel particulate matter; hPSC, human pluripotent stem cell; AEC, alveolar epithelial cells.

Fig. 4.. Effects of nicotine exposure on cell death, inflammation and oxidative stress in hPSC-AECs.

hPSC-AECs were treated with the indicated concentrations of nicotine for 48 hrs. Transcript levels of cell death (A), inflammation (B) and oxidative stress (C)-related genes were measured by quantitative real-time PCR. Data expressed a mean±SD. * p<0.05, ** p<0.01 vs Control. hPSC, human pluripotent stem cell; AEC, alveolar epithelial cells.

Fig. 5.. Effects of CSE exposure on cell death, inflammation and oxidative stress in hPSC-AECs.

hPSC-AECs were treated with the indicated concentrations of CSE for 48 hrs. Transcript levels of cell death (A), inflammation (B) and oxidative stress (C)-related genes were measured by quantitative real-time PCR. Data expressed a mean±SD. * p<0.05, ** p<0.01 vs Control. CSE, cigarette smoke extract; hPSC, human pluripotent stem cell; AEC, alveolar epithelial cells.