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Single-Cell RNA-Sequencing of Human Embryonic Stem Cell Differentiation Delineates Adverse Effects of Nicotine on Embryonic Development

ABSTRACT: Nicotine, the main constituent of tobacco, is highly detrimental to the developing fetus by increasing the risk of gestational complications and organ disorders. The effects of nicotine on human embryonic development and related mechanisms, however, remain largely unresolved. Here, we performed single-cell RNA-sequencing (scRNA-seq) of human embryonic stem cell (hESC)-derived embryoid body (EB) in the presence or absence of nicotine. Nicotine induced lineage-specific responses and dysregulated cell-to-cell communication in EBs, shedding light on the adverse effects of nicotine on human embryonic development. Additionally, nicotine reduced cell viability, increased reactive oxygen species (ROS), and altered cell cycling in EBs. Abnormal Ca2+ signaling was found in muscle cells upon nicotine exposure and verified in hESC-derived cardiomyocytes. Consequently, our scRNA-seq data suggests direct adverse effects of nicotine on hESC differentiation at the single-cell level and offers new method for evaluating drug and environmental toxicity on human embryonic development in utero.

ORGANISM(S): Homo sapiens

PROVIDER: GSE125416 | GEO | 2019/01/22

REPOSITORIES: GEO

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Project description:Directing differentiation of human embryonic stem cells (hESC) into specific cell types using an easy and reproducible protocol is a perquisite for the clinical use of hESC in regenerative medicine protocols. Here, we report the generation of mesodermal cells with differentiation potential to myocytes, osteoblasts, chondrocytes and adipocytes. We demonstrate that during hESC differentiation as embryoid bodies (EB), inhibition of TGF-b/Activin/Nodal signaling using SB-431542 (SB) markedly up-regulated paraxial mesodermal markers (TBX6, TBX5), early myogenic transcriptional factors (Myf5, Pax7) as well as myocyte committed markers (NCAM, CD34, Desmin, MHC (fast), alpha-smooth muscle actin, Nkx2.5, cTNT). Establishing EB outgrowth cultures (SB-OG) in the presence of SB (1 uM) led to further enrichment of cells expressing markers for myocyte progenitor cell: CD34+ (33%), NCAM+ (CD56) (73%), PAX7 (25%) and mature myocyte proteins (MYOD1, tropomyocin, fast MHC an; d SERCA1). Further analysis using DNA microarray revealed differential up-regulation of 117 genes (>2-fold compared to control cells) annotated to myogenic development and function. During ex vivo culture, contracting myocytes were observed (80% of the population) and the cells formed myofibres when implanted intramuscularly in vivo. Furthermore, in the presence of fetal bovine serum (10% FBS), SB-OG cells developed morphologically and phenotypically into a homogeneous stromal (mesenchymal) stem cell (MSC)-like population expressing characteristic MSC CD markers: CD44 (100%), CD73 (98%), CD146 (96%) and CD166 (88%). They were karyotypically normal and were able to differentiate ex vivo and in vivo into osteoblasts, adipocytes and chondrocytes. Experiment Overall Design: Human ESCs were differentiated as EBs for 10 days, where after EBs were plated onto fibronectin coated petri dishes. At confluency the outgrowth culture was passaged, at passage 5 3 samples were taken from both control-OG and SB-OG. the samples were mixed and frozen.

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Single-Cell RNA-Sequencing of Human Embryonic Stem Cell Differentiation Delineates Adverse Effects of Nicotine on Embryonic Development

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