Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Nov;116(11):e2409.
doi: 10.1002/bdr2.2409.

A Single Cell Transcriptomic Fingerprint of Stressed Premature, Imbalanced Differentiation of Embryonic Stem Cells

Ximena L Ruden  1   2 , Aditi Singh  1   3 , Teya Marben  4 , Wen Tang  5 , Awoniyi O Awonuga  1 , Douglas M Ruden  1   6 , Elizabeth E Puscheck  1   2   7 , Hao Feng  5 , Steven J Korzeniewski  8 , Daniel A Rappolee  1   2   3   9
Affiliations

A Single Cell Transcriptomic Fingerprint of Stressed Premature, Imbalanced Differentiation of Embryonic Stem Cells

Ximena L Ruden et al. Birth Defects Res. 2024 Nov.

Abstract

Background: Miscarriages cause a greater loss-of-life than cardiovascular diseases, but knowledge about environmentally induced miscarriages is limited. Cultured naïve pluripotent embryonic stem cells (ESC) differentiate into extra-embryonic endoderm/extraembryonic endoderm (XEN) or formative pluripotent ESC, during the period emulating maximal miscarriage of peri-implantation development. In previous reports using small marker sets, hyperosmotic sorbitol, or retinoic acid (RA) decreased naïve pluripotency and increased XEN by FACS quantitation.

Methods: Bulk and single cell (sc)RNAseq analyses of two cultured ESC lines was done, corroborated by qPCR. Transcriptomic responses were analyzed of cultured ESC stressed by Sorbitol, with Leukemia inhibitory factor (LIF + ; stemness growth factor), RA without LIF to control for XEN induction, and compared with normal differentiation (LIF - , ND).

Results: Sorbitol and RA increase subpopulations of 2-cell embryo-like (2CEL) and XEN sub-lineages; primitive, parietal, and visceral endoderm (VE) cells and suppress formative pluripotency, imbalancing alternate lineage choices of initial naïve pluripotent cultured ESC compared with ND. Although bulk RNAseq and gene ontology (GO) group analyses suggest that stress induces anterior VE-head organizer and placental markers, scRNAseq reveals relatively few cells. But VE and placental markers/cells were in adjacent stressed cell clusters in the UMAP, like recent, normal UMAP of conceptuses. UMAPs show that dose-dependent stress overrides stemness to force premature lineage imbalance.

Conclusions: Hyperosmotic stress, and other toxicological stresses, like drugs with active ingredient RA, may cause premature, lineage imbalance, resulting in miscarriages or birth defects.

Keywords: XEN lineages; compensatory differentiation; embryonic stem cells; high throughput; hyperosmotic stress; pluripotency; transcription factors.

PubMed Disclaimer

Update of

References

    1. Abdulhasan, M., X. Ruden, T. Marben, et al. 2022. “Using Live Imaging and Fluorescence Ubiquitinated Cell Cycle Indicator Embryonic Stem Cells to Distinguish G1 Cell Cycle Delays for General Stressors Like Perfluoro‐Octanoic Acid and Hyperosmotic Sorbitol or G2 Cell Cycle Delay for Mutagenic Stressors Like Benzo(a)pyrene.” Stem Cells and Development 31, no. 11–12: 296–310.
    1. Abdulhasan, M., X. Ruden, B. Rappolee, et al. 2021. “Stress Decreases Host Viral Resistance and Increases Covid Susceptibility in Embryonic Stem Cells.” Stem Cell Reviews and Reports 17, no. 6: 2164–2177.
    1. Anders, S., P. T. Pyl, and W. Huber. 2015. “HTSeq—A Python Framework to Work With High‐Throughput Sequencing Data.” Bioinformatics 31, no. 2: 166–169.
    1. Andrews, S. 2010. “FastQC A Quality Control Tool for High Throughput Sequence Data.”
    1. Artus, J., J.‐J. Panthier, and A.‐K. Hadjantonakis. 2010. “A Role for PDGF Signaling in Expansion of the Extra‐Embryonic Endoderm Lineage of the Mouse Blastocyst.” Development 137, no. 20: 3361–3372.

MeSH terms

LinkOut - more resources