It is well recognized that maternal smoking during pregnancy is a risk factor for miscarriage, low birthweight, and premature birth. Researchers at Stanford University School of Medicine have now developed a human embryonic stem cell (hESC) model to demonstrate how nicotine—which is also found in e-cigarette liquid and smoking cessation aids—adversely affects different cell types in the developing embryo.

The authors, led by Joseph C. Wu, MD, PhD, director of the Stanford Cardiovascular Institute and Simon H. Stertzer endowed professor in the departments of medicine and radiology at the Stanford School of Medicine, suggest that their model could offer new insights into the effects of nicotine on individual organs and cells within the developing fetus, and may also be used to optimize drug and environmental toxicity screening.

“Nicotine found in products such as tobacco, e-cigarettes, and nicotine gums may have wide-ranging, harmful effects on different organs of a developing embryo during pregnancy,” commented Wu. “These results are especially important in that they provide a scientific basis for educating the public, especially young women, to keep away from smoking when they are pregnant or considering having a family.”

Wu’s team, working with colleagues at the Stanford Cardiovascular Institute, the Institute for Stem Cell Biology and Regenerative Medicine, Division of Cardiology, and at the University of Arizona College of Medicine, reported their findings in Stem Cell Reports, in a paper titled, “Single-Cell Sequencing of Human Embryonic Stem Cell Differentiation Delineates Adverse Effects of Nicotine on Embryonic Development.”

Maternal smoking during pregnancy is a known risk for birth defects, but is also closely linked with adverse neurobehavioural, cardiovascular, respiratory, endocrine, and metabolic outcomes, which can last into adulthood, the authors explained. Nicotine, the main chemical component of tobacco smoking, is primarily responsible for this elevated risk. “Unfortunately,” they noted, “the introduction and spread of new tobacco products containing nicotine, such as e-cigarettes, is reversing recent progress toward reduction of tobacco use.”

The adverse effects of nicotine in animals—primarily rodents—have been well researched, and demonstrated that exposure to the drug during pregnancy has detrimental effects on aspects of fetal development including cellular damage, increased inflammation, oxidative stress, and impaired cell replication. However, carrying out such detailed investigations in humans isn’t feasible. “The effects of nicotine on human embryonic development and related mechanisms, however, remain poorly understood,” the researchers commented.

To address some of the limitations, Wu’s team and their collaborators used single-cell RNA sequencing (scRNA-seq) to analyze the effects of 21 days of nicotine exposure on the transcriptomes of 12,500 cells generated from human embryonic stem cell-derived embryoid bodies (EBs). These are laboratory-grown 3D aggregates of different types of pluripotent cells that will differentiate into the variety of cell types that ultimately form organs including the heart, brain, liver, blood vessels, and muscles. While the use of in vitro-differentiated embryonic bodies to model early embryonic development isn’t new, previous work looking at gene expression has harnessed conventional, bulk RNA sequencing, which has limited utility for studying individual cell types.

The advent of microdroplet-based scRNA-seq technology means it is now possible to analyze transcripomes at the single cell level, within heterogeneous cell populations. “Here, we used scRNA-seq of EBs to characterize the effects of nicotine on hESC differentiation,” the authors commented. “Integrated analysis of control and nicotine-exposed EBs at the single-cell level enables us to quantitatively assess cell-type-specific responses to nicotine.”

This image illustrates how researchers studied the adverse effects of nicotine exposure on various cell lineages derived from human embryonic stem cells using single-cell RNA sequencing (scRNA-seq). [Hongchao Guo]
The EB cell types analyzed included six major progenitors: neural, liver, stromal, endothelial, epithelial, and muscle. The results broadly showed that exposure to nicotine reduced cell viability, increased reactive oxygen species (ROS), and decreased the size of the embryoid bodies, resulting in aberrant formation and differentiation. Nicotine exposure also altered cell cycling in various progenitor cells differentiated from hESCs, and caused dysregulated cell-to-cell communication, an adverse effect that hasn’t yet been well studied.

Interestingly, quantification of the cell-type compositional changes showed that nicotine exposure was associated with a 5% reduction in epithelial cells, and a 4% increase in liver cells. There was also considerable difference in the relative proportion of genes that were affected by nicotine in each cell type, with muscle cells exhibiting the greatest number of differentially expressed genes.

The scRNA-seq results showed cell type-specific changes in the expression of genes implicated in metal toxicity and mitochondrial function, brain malformations and intellectual disability, muscle development and disease, lung disease, and Ca2+-associated arrhythmias that affect the contractility of heart muscle cells.

In neural progenitor cells, for example, nicotine exposure resulted in the up- or downregulation of certain genes, the abnormal expression of which is already known to lead to β-amyloid formation and increased synaptic transmission, brain malformations, and intellectual disability. In muscle cells, the most upregulated gene in response to nicotine exposure was a myosin chaperone protein gene that is involved in muscle development and disease.

In stromal cells, two genes known to regulate nutrient levels and amino acid acetylation were downregulated following nicotine exposure, while liver cells demonstrated downregulation of a gene related to lipid metabolism. Notable in epithelial cells was reduced expression of a gene associated with chronic obstructive pulmonary disease, while there were cell-type-specific alterations in the expression of genes in endothelial cells related to suppression of glycolysis and endothelial cell dysfunction, and endothelial lumen formation during angiogenesis.

The DEG patterns from the various progenitor cell populations indicated broad effects on cells derived from all three germ layers. “This is important because we know that smoking and nicotine have been shown to increase the pathological risk in endocrine, reproductive, respiratory, cardiovascular, and neurologic systems that rely on intricate and dynamic interactions amongst multiple cell types for homeostasis and function,” Wu added.

“Our study offers an effective platform to evaluate the potential effects of nicotine on human embryonic development,” the team concluded. “Our data provide potential molecular mechanisms for prenatal nicotine toxicity on specific cell populations derived from human ESCs.”

“A major implication of our study is that we now have validated a new method for evaluating the effect of drugs and environmental toxicity on human embryonic development,” Wu said. He acknowledged that one limitation of the embryoid body model is that it can’t recapitulate the whole-body physiology of a pregnant woman. “For example, the influence of exercise, stress, food, or hormonal changes are not captured in this model,” he stated.

The researchers aim to continue investigating the mechanisms of nicotine-induced fetal birth defects. “We hope this will lead to the discovery of novel biomarkers that can help doctors better prevent, diagnose, and treat these diseases,” Wu noted. “In addition, we plan to utilize our hESC-derived embryoid body model and single-cell-sequencing technology to investigate the wider effects of other harmful conditions such as air pollution on human embryonic development.”

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