Nicotine-induced Genetic and Epigenetic Modifications in Primary Human Amniotic Fluid Stem Cells

Abstract

Background: Smoking during pregnancy has been linked to adverse health outcomes in offspring, but the underlying mechanisms are not fully understood. To date, the effect of maternal smoking has been tested in primary tissues and animal models, but the scarcity of human tissues limits experimental studies. Evidence regarding smoking-related molecular alteration and gene expression profiles in stem cells is still lacking.

Methods: We developed a cell culture model of human amniotic fluid stem cells (hAFSCs) of nicotine (NIC) exposure to examine the impact of maternal smoking on epigenetic alterations of the fetus.

Results: NIC 0.1 μM (equivalent to "light" smoking, i.e., 5 cigarettes/day) did not significantly affect cell viability; however, significant alterations in DNA methylation and N6-methyladenosine (m6A) RNA methylation in hAFSCs occurred. These epigenetic changes may influence the gene expression and function of hAFSCs. Furthermore, NIC exposure caused time-dependent alterations of the expression of pluripotency genes and cell surface markers, suggesting enhanced cell stemness and impaired differentiation potential. Furthermore, NICtreated cells showed reduced mRNA levels of key adipogenic markers and hypomethylation of the promoter region of the imprinted gene H19 during adipogenic differentiation, potentially suppressing adipo/lipogenesis. Differential expression of 16 miRNAs, with predicted target genes involved in various metabolic pathways and linked to pathological conditions, including cognitive delay and fetal growth retardation, has been detected.

Conclusion: Our findings highlight multi-level effects of NIC on hAFSCs, including epigenetic modifications, altered gene expression, and impaired cellular differentiation, which may contribute to long-term consequences of smoking in pregnancy and its potential impact on offspring health and development.

Keywords: Primary human amniotic fluid stem cells; adipogenesis; epigenetics; mRNAs.; nicotine; tobacco-related disorders.

Fig. (1)

The evaluation of cell viability in hAFSCs after treatment with Nicotine (NIC). hAFSCs (3000 cells/well) were incubated for 24 hours at 37°C in a humidified atmosphere (95%) under 5% CO₂. Then, cells were treated with NIC 0.1 μM and incubated at 37°C in a humidified atmosphere with 5% CO₂ for 24, 48, and 72 hours respectively. At each time point, the cell viability was assessed. Background absorbance (490 nm) was subtracted from each data point using a set of wells containing only Iscove's Modified Dulbecco's Medium (IMDM). Data are reported as mean ± SD n = at least 6 (at each time point); “ns” indicates not significant (P > 0.05).

Fig. (2)

(A) Effects of nicotine (NIC) treatment on global DNA methylation (5 m-C) of hAFSCs (undifferentiated). hAFSCs (3000 cells/well) were incubated for 24 hours at 37°C in a humidified atmosphere (95%) under 5% CO₂. Then, the cells were treated with Nicotine 0.1 μM and incubated at 37°C in a humidified atmosphere with 5% CO₂ for 6, 24, and 48 hours respectively. Global DNA methylation quantification was assessed and reported, at each time point, as 5 mC/C ratios (%). At each time point, data are reported as mean ± SD, n=3 (each analyzed in duplicate). *** ≤ 0.001 and **** ≤ 0.0001 vs. control (untreated cells) at the same time point. (B) The effects of Nicotine (NIC) treatment on N6-methyladenine (m⁶A) modification of hAFSCs (undifferentiated). hAFSCs (3000 cells/well) were incubated for 24 hours at 37°C in a humidified atmosphere (95%) under 5% CO₂. Then, the cells were treated with NIC 0.1 μM and incubated at 37°C in a humidified atmosphere with 5% CO₂ for 6, 24, and 48 hours, respectively. m⁶A methylation was assessed as m⁶A/A ratios (%). Data are reported as mean ± SD, n = 3 (each analyzed in duplicate). ***P ≤ 0.001 and ****P ≤ 0.0001 vs. control (untreated cells) at the same time point.

Fig. (3)

The effects of nicotine (NIC) treatment on the expression of pluripotency markers of hAFSCs (undifferentiated). hAFSCs (3000 cells/well) were incubated for 24 hours at 37°C in a humidified atmosphere (95%) under 5% CO₂. Then, the cells were treated with NIC 0.1 μM and incubated at 37°C in a humidified atmosphere with 5% CO₂ for 6, 24, and 48 hours respectively. The gene expression of Oct-4 (POU class 5 homeobox 1), SOX2 (SRY-box transcription factor 2), NANOG (Nanog homeobox), and C-Kit (KIT proto-oncogene, receptor tyrosine kinase) was evaluated by qPCR and normalized to those of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and reported as fold-change vs. the gene expression detected in control samples. Data are reported as mean ± SD, n = 5 (each analyzed in duplicate) *P ≤ 0.05, **P ≤ 0.01; and ****P ≤ 0.0001 vs. control (untreated cells) at the same time point.

Fig. (4)

(A) The effects of NIC treatment on the expression of adipogenic markers in hAFSCs (differentiated). In addition, to induce adipogenic differentiation, once hAFSCs have reached the confluence of 90%, the medium was replaced with the adipogenic differentiation medium (AdipoMAX Differentiation Medium). The cells were left to differentiate for 21 days in the absence (control) or in the presence of Nicotine (NIC) 0.1 μM (treated) for the same time period. The gene expression of LPL (lipoprotein lipase), PPARG (peroxisome proliferator-activated receptor gamma), and FABP4 (fatty acid binding protein 4) was evaluated by qPCR and normalized to those of GAPDH and reported as fold-change vs. the gene expression detected in control samples. Data are reported as mean ± SD, n=3 (each analyzed in duplicate). ****P ≤ 0.0001 vs. control (untreated cells). (B) Promoter-specific methylation change in control (untreated), differentiated nicotine (NIC) 0.1 µM treated (21 days), and untreated cells (21 days). Data are reported as mean ± SD n = 3 (each analyzed in duplicate). ****P ≤ 0.0001 vs. hAFSCs (undifferentiated) and # P ≤ 0.0001 vs. hAFSCs (untreated).

Fig. (5)

(A and B) Significant changes of miRNA expression in NIC 0.1 µM treated cells (differentiated) for 21 days by using Next Generation approach (Illumina platform). Fold change ≥ 2 are included in the study (n=5). (C) Pathway prediction of miRNAs differentially expressed between treated differentiated hAFSCs and untreated differentiated cells. Red colour shows the most significant pathway involving each miRNA.