The Developmental Toxicity And Mechanism Of PM2.5 On The Early Stages Of Fetal Lung With Human Lung Bud Tip Progenitor Organoids

Fine particulate matter(PM2.5),as a major component of atmospheric pollutants,has become one of the main risk factors for environmental health.Much attention has been focused on the respiratory health of PM2.5.Epidemiological studies have shown that exposure to PM2.5 during pregnancy can affect fetal lung development,leading to neonatal lung function impairment.However,due to the lack of reliable human lung development models and ethical issues,the effects and mechanisms of PM2.5 on lung development,especially for the early stages of fetal lung development,are not fully understood.In this study,the lung bud tip progenitor organoids(LPOs)were established using human embryonic stem cells(hESCs)line H9 to mimick the early stages of fetal lung development.And the potential lung developmental toxicity of PM2.5 was assessed based on the LPOs model.The results of our study are detailed as follows:1.Generation of hESC-derived LPOs:hESCs highly expressed of pluripotencyrelated markers OCT-4 and NANOG were induced to differentiate into DE with the expression of SOX17+/FOXA2+by small molecule Activin A treatment.Following formation of the SOX2+positive AFE.Finally,LPOs were successfully established from hESCs,and confirmed that the organoids were positive for the lung progenitor cell markers NKX2.1,SOX2 and SOX9.2.The developmental toxicity of PM2.5 on the hESCs-derived LPOs:It demonstrated that PM2.5 with concentrations of 12.5 and 25 mg/l did not cause cytotoxicity and did not affect stemness gene expression up to 72 h for hESCs.Therefore,we selected these two concentrations of PM2.5 to further investigate the effects on early fetal lung development.The whole procedure from induction of hESCs to LPOs was continuous exposure to PM2.5.At the end of treatment,the proliferation ability of LPOs was significantly decreased by 30%.At the same time,the gene expression levels of the key transcription factor SOX2 for proximal lung differentiation and the lung endoderm marker NKX2.1 decreased by 25%and 30%,respectively.After 12.5 mg/l PM2.5 treatment,the gene expression level of the key transcription factor SOX9 for distal differentiation was upregulated by 50%.Meanwhile,Wnt/β-catenin signaling pathway,which plays a major role in fetal lung development,was activated.And the downstream target genes AXIN2,β-catenin,and TCF/LEF were significantly upregulated by 50%,20%,and 33%,respectively.These results suggest that PM2.5 exposure may disturb the balance between proximal and distal lung development through Wnt/β-catenin signaling pathway.3.Effects of PM2.5 on different stages of hESCs differentiation into LPOs:To further explore the critical sensitive period of PM2.5 exposure during the process of forming LPOs from hESCs,we investigated the effects of PM2.5 exposure on the formation of DE and AFE,respectively.We found that PM2.5 exposure significantly inhibited the differentiation of hESCs into DE.After treatment with 12.5 mg/l PM2.5,the gene expression level of the key marker SOX17 decreased by 64%.After treatment with 25 mg/l PM2.5,the gene expression levels of SOX17 and FOXA2 decreased by 65%and 60%,respectively,and the protein expression levels of SOX 17 and FOXA2 decreased by 67%and 36%,respectively.In addition,the expression levels of AXIN2 and β-catenin,the target genes of Wnt/β-catenin signaling pathway,were downregulated by 40%and 45%,respectively,after 12.5 mg/l PM2.5 treatment.25 mg/l PM2.5 treatment downregulated the expression levels of AXIN2,β-catenin and TCF/LEF by 67%,71%and 38%.PM2.5 exposure of DE induced differentiation to AFE stage revealed that cell viability of AFE decreased by 39%and AFE spheroid formation efficiency was significantly reduced after 25 mg/l PM2.5 treatment.Gene expression levels of NKX2.1,a key marker of AFE differentiation,decreased by 28%and 25%,respectively,after PM2.5 treatment.However,it did not affect the expression of the marker SOX2 gene.In addition,the expression levels of AXIN2,β-catenin and TCF/LEF,the target genes of Wnt/β-catenin signaling pathway,were down-regulated by 30%,41%and 24%,respectively,after 25 mg/l PM2.5 treatment.The above results suggest that Wnt/β-catenin signaling pathway is involved in the toxicity of PM2.5 to DE and AFE.In summary,we provided initial evidence that PM2.5 could affect the proximaldistal differentiation of fetal lung development through Wnt/β-catenin signaling pathway using LPOs model.The results of this study revealed the potential health risks of prenatal exposure to PM2.5 on respiratory system development in offspring.