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

IF 1.6 4区医学 Q4 DEVELOPMENTAL BIOLOGY

Birth Defects Research Pub Date : 2024-10-31 DOI:10.1002/bdr2.2409

Ximena L Ruden, Aditi Singh, Teya Marben, Wen Tang, Awoniyi O Awonuga, Douglas M. Ruden, Elizabeth E Puscheck, Hao Feng, Steven J. Korzeniewski, Daniel A Rappolee

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引用次数: 0

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.

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胚胎干细胞受压过早失衡分化的单细胞转录组指纹图谱

背景：与心血管疾病相比，流产造成的生命损失更大，但有关环境诱发流产的知识却很有限。培养的幼稚多能胚胎干细胞（ESC）在胚胎围着植入发育的最大流产模拟期分化为胚外内胚层/胚外内胚层（XEN）或形成性多能胚胎干细胞。在之前使用小标记集的报告中，通过 FACS 定量，高渗山梨醇或维甲酸（RA）降低了幼稚多能性，增加了 XEN：方法：对两个培养的 ESC 株系进行了大量和单细胞（sc）RNAseq 分析，并通过 qPCR 进行了证实。分析了受山梨醇、白血病抑制因子（LIF +；干性生长因子）和不含 LIF 的 RA 胁迫的培养 ESC 的转录组反应，以控制 XEN 诱导，并与正常分化（LIF - , ND）进行比较：结果：与ND相比，山梨醇和RA增加了2细胞胚胎样（2CEL）和XEN亚系、原始细胞、顶叶细胞和内脏内胚层（VE）细胞的亚群，并抑制了形成性多能性，使初始幼稚多能培养ESC的交替系选择失衡。虽然大量 RNAseq 和基因本体（GO）组分析表明应激会诱导前 VE 头组织者和胎盘标记，但 scRNAseq 发现的细胞相对较少。但在UMAP中，VE和胎盘标记/细胞位于相邻的受压细胞簇中，就像最近的正常概念胎的UMAP一样。UMAPs表明，剂量依赖性应激超越了干性，迫使过早的系失衡：结论：高渗应激和其他毒性应激，如含有活性成分RA的药物，可能会导致过早的细胞系失衡，从而导致流产或出生缺陷。

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来源期刊

Birth Defects Research Medicine-Embryology

CiteScore

3.60

自引率

9.50%

发文量

153

期刊介绍： The journal Birth Defects Research publishes original research and reviews in areas related to the etiology of adverse developmental and reproductive outcome. In particular the journal is devoted to the publication of original scientific research that contributes to the understanding of the biology of embryonic development and the prenatal causative factors and mechanisms leading to adverse pregnancy outcomes, namely structural and functional birth defects, pregnancy loss, postnatal functional defects in the human population, and to the identification of prenatal factors and biological mechanisms that reduce these risks. Adverse reproductive and developmental outcomes may have genetic, environmental, nutritional or epigenetic causes. Accordingly, the journal Birth Defects Research takes an integrated, multidisciplinary approach in its organization and publication strategy. The journal Birth Defects Research contains separate sections for clinical and molecular teratology, developmental and reproductive toxicology, and reviews in developmental biology to acknowledge and accommodate the integrative nature of research in this field. Each section has a dedicated editor who is a leader in his/her field and who has full editorial authority in his/her area.