Impairment And Mechanisms In Stemness Of Human Mesenchymal Stem Cells Exposed To Alternatives Of Perfluorinated Compounds

More than 3000 per-and polyfluoroalkyl substances(PFASs)are in use globally,for which the environmental effects and health risks are not well-known,challenging the regulation of PFASs broadly as a chemical class.In the present study,human bone mesenchymal stem cells(hBMSCs)were used as an in vitro model to evaluate the toxicity and potential molecular mechanisms of 3 substitutes for PFASs including chlorinated polyfluorinated ether sulfonate(Cl-PFESA),hexafluoropropylene oxide dimer acid(HFPO-DA)and hexafluoropropylene oxide trimer acid(HFPO-TA)on stemness of hBMSCs.The main contents and results include,(1)Gene profiles of which affected by Cl-PFESA,perfluorooctane sulfonate(PFOS),perfluorohexane sulfonate(PFHxS),and perfluorooctanoic acid(PFOA)were analysed by weighted gene co-expression network analysis(WGCNA)to reveal the toxic targets of PFASs.Correlation analysis between the modules and cell samples indicated that gene profiles in control cells positively correlated with Blue and Greenyellow modules.Gene profiles in cell cultures treated by PFOS negatively correlated with Blue module and that treated by Cl-PFESA exposure negatively correlated with Greenyellow module,which suggested different gene expression in Blue and Greenyellow module between vehicle and PFASs exposure.The most significantly enriched biological processes in Blue and Greenyellow modules were cholesterol biosynthetic process and negative regulation of myeloid leukocyte differentiation,respectively,implied that immunoregulation and lipid metabolism were potential targets of PFASs.DHCR24,SQLE and EBP,key genes involved in lipid metabolism,might be target genes of PFASs.(2)Human bone mesenchymal stem cells(hBMSCs)were used to evaluate the effects of Cl-PFESA at non-cytotoxic concentrations on molecular regulation and cellular function of stem cells compared to PFOS,PFHxS and PFOA.Gene profiles of hBMSCs exposed to 100 nM of Cl-PFESA and the other 3 perfluoroalkyl acids(PFAAs)correlated significantly with each other.A total of 261 genes were found to be affected by all 4 compounds.Functional annotation analysis revealed that osteoblast differentiation,ERK1/2,TGF? and calcium signalling were interfered.Moreover,DUSP mRNA and P-SMAD protein,key factors in ERK and TGF?/SMAD signaling,were decreased by Cl-PFESA.Furthermore,intracellular calcium image suggested that calcium transients were enhanced by Cl-PFESA with lower effective concentrations and more prolonged induction than PFOS and PFHxS.Immunization staining confirmed that the stemness marker CD44 was dose-dependently repressed by Cl-PFESA.In the osteogenic differentiation following exposure to 100 nM of Cl-PFESA,both mRNA and protein of RUNX2,a target of multiple osteogenic pathways,was depressed on differentiation day 7.Exposure to Cl-PFESA at human relevant concentrations during a vulnerable period before differentiation posed persistent effects on hBMSCs,with common or even stronger potency compared to PFAAs.(3)Toxic effects of perfluoroalkyl ether carboxylic acids(PFECAs)were investigated.After 14 and 21 days of exposure,HFPO-DA show cytotoxicity on hBMSCs.Stemness markers of hBMSCs were inhibited significantly in mRNA and protein level by HFPO-DA and HFPO-DA exposure,which suggested that stemness of hBMSCs was impaired.Both DAVID and IPA significantly enriched pathways related to pluripotency of stem cells and carcinogenesis.Transcriptome analysis further indicated that pluripotency of hBMSCs was affected,and genes associated with enriched cancer pathway may be target genes for HFPO-DA and HFPO-DA.TP53,identified by upstream analysis of IPA,may be potential target proteins for HFPO-DA and HFPO-DA.Finally,the accumulation of fat droplets was promoted by HFPO-DA and HFPO-DA exposure,which suggested that HFPO-DA and HFPO-DA may also interfere with the differentiation process of hBMSCs.The present study employed in vitro hBMSCs model and comparative transcriptome analysis to reveal the effect and potential mechanisms of PFASs substitutes on the stemness of hBMSCs,which would benefit the development of biomarkers for toxicity prediction and screening of PFASs,as well as provide experimental evidence and novel method for the health risk assessment and regulation.