2. Academic Validation
  3. The role of ASS1 in mouse embryonic stem cell differentiation into mesendoderm lineages

The role of ASS1 in mouse embryonic stem cell differentiation into mesendoderm lineages

  • Stem Cell Res Ther. 2025 Sep 1;16(1):479. doi: 10.1186/s13287-025-04622-w.
Guodong Zhao 1 2,  Qiaoqiao Meng 1 2,  Shuo Cao 1 2,  Qiqi Jiang 1 2,  Yanglin Chen 1 2,  Baojiang Wu 1 2,  Xihe Li 3 4 5,  Siqin Bao 6 7
Affiliations

Affiliations

  • 1 Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, 010020, China.
  • 2 The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, 010020, China.
  • 3 Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, 010020, China. lixh@imu.edu.cn.
  • 4 The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, 010020, China. lixh@imu.edu.cn.
  • 5 Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, 011517, China. lixh@imu.edu.cn.
  • 6 Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, 010020, China. baosq@imu.edu.cn.
  • 7 The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, 010020, China. baosq@imu.edu.cn.
PMID: 40890876 DOI: 10.1186/s13287-025-04622-w
Abstract

Background: Argininosuccinate synthase (ASS1) is a pivotal enzyme involved in the urea cycle, playing a crucial role in aspartate catabolism, arginine and nitric oxide biosynthesis. These biological processes are crucial for the growth and development of mammals. However, the functions of urea cycle-related genes in mouse embryonic stem cells (mESCs) remain largely unclear. Here, we investigated the impact of ASS1 knockout on the mESCs pluripotency and its role in determining cell fate.

Methods: ASS1 was knocked out in mESCs using CRISPR-Cas9. Changes in pluripotency post-knockout were analyzed via immunofluorescence, Western blotting, differentiation assays, and chimera formation. Cardiomyocyte differentiation assays evaluated the impact on cardiac lineage specification. RNA Sequencing (RNA-seq), Western blotting, and signaling pathway inhibitors were used to investigate alterations in molecular signatures and regulatory mechanisms.

Results: ASS1 knockout did not compromise mESCs pluripotency maintenance or self-renewal but perturbed the cell cycle. It upregulated mesendoderm markers while downregulating ectoderm markers. Notably, ASS1 deficiency enhanced cardiomyocyte differentiation potential. The multi-lineage differentiation markers were reversed by either ASS1 overexpression or treatment with Wnt/β-catenin inhibitors.

Conclusions: ASS1 knockout directs mESCs toward mesendoderm lineage commitment, thereby promoting beating cardiomyocyte generation. Thus, ASS1 acts as a regulatory switch whose absence drives mesendoderm differentiation and enhances cardiomyocyte production.

Keywords

Argininosuccinate synthase (ASS1); Cardiomyocyte; Mesendoderm; Mouse embryonic stem cells (mESCs); Wnt/β-catenin.

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