Journal of Integrative Agriculture

• •    下一篇

TBX3推动猪iPSC从多能态向类全能态的转变

  

  • 修回日期:2024-03-07

TBX3 orchestrates H3K4 trimethylation for porcine induced pluripotent stem cells to totipotent-like stem cells

Qiaoyan Shen1, 7, Xiaojie Wu1, Ziyu Chen2, Jianxiong Guo4, Wei Yue1, Shuai Yu1, Rui Zhang1, Xiaolong Wu1, Heng Zhao4, Qin Pan1, Juqing Zhang1, Zhenshuo Zhu1, Xinchun Yang1, Wenjing Xu1, Yunxiang Li1, Anmin Lei1, Sha Peng1, Fan Yang1, Shiqiang Zhang1, Gang Ren5, Jun Wu6, Na Li1*, Hongjiang Wei3,4*, Mingzhi Liao2*, Jinlian Hua1*   

  1. 1 College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling 712100, Shaanxi, China;

    2 College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, China.

    3 State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China.

    4 College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.

    5 College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.

    6 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    7  State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. University of Chinese Academy of Sciences, Beijing 100049, China.

  • Revised:2024-03-07
  • About author:Qiaoyan Shen, E-mail:sqy@nwafu.edu.cn; Xiaojie Wu, E-mail:wuxiaojie@ nwafu.edu.cn; #Correspondence Jinlian Hua, E-mail: jinlianhua@nwsuaf.edu.cn; Mingzhi Liao, E-mail: liaomz@nwsuaf.edu.cn; Hongjiang Wei, E-mail: hongjiang-wei@126.com; Na Li, E-mail: lina2017@nwafu.edu.cn

PDF

摘要： 目前人们所获得的猪多能干细胞普遍缺乏有效嵌合能力，极大的阻碍了猪发育生物学及干细胞育种等领域的研究进展。本研究通过 TBX3 导入 OSKM（OCT4、SOX2、KLF4、c-MYC）诱导所建立的猪诱导多能干细胞（induced Pluripotent Stem Cells，iPSCs），发现 OSKMT 细胞相对 OSKM 细胞具有胚内与胚外谱系分化的全胚层发育能力，具有全能性细胞的特征totipotent-like stem cells (TLSCs)。主要实验结果如下：1.OSKMT 细胞株整体转录水平与猪 4 细胞期胚胎相接近。通过OSKM 细胞株与 OSKMT细胞株的构建，证实TBX3 有利于猪 iPSCs 重编程的加速进行。同时，且OSKMT细胞株呈现出广泛的4 细胞胚胎期基因 MCL1、PDIA3、SYT5等基因相对上调表达的特征。2.体外类囊胚 （Blastoids）的形成确认 OSKMT 细胞株在体外具有全谱系分化能力：通过拟胚体（Embryoid Bodies，EBs）与畸胎瘤的形成，发现 OSKMT 具有分化产生滋养层与体内三胚层组织细胞的特性。单细胞源人造 Blastoids 的形成是评价全能性的重要指标，在 EBs 的形成中，OSKMT 细胞具有自发形成 Blastoids 的能力，进一步确认了 OSKMT 细胞的全能性。3.体内嵌合实验确认 OSKMT 细胞株的类全能性：对 OSKMT 细胞株进行嵌合能力的分析，证明其在小鼠早期胚胎的异种嵌合中具有 TE 与 ICM 的双向嵌合特征与更高的存活率，并获得了发育 28 天的胎盘与胎儿双嵌合特征的嵌合体，证明了OSKMT 细胞具有全能性。4.TBX3 激活 H3K4me3 甲基转移酶 MLL1 的转录，进而催化 H3K4me3 修饰的转变：解析 TBX3 的作用机制，发现 TBX3 的表达有利于 MLL1，SETD1A 等 H3K4me3 甲基转移酶转录激活，发现 MLL1 对滋养层发育能力存在显著性影响，揭示 MLL1 是全能性获得的重要靶点。5.MCL1 是TBX3 - MLL1调控轴的重要靶点：寻找 TBX3 - MLL1 调控轴的下游靶基因。MCL1 是猪 ZGA 时期特征性基因。在 OSKMT 组中 H3K4me3在 MCL1 的 TSS 区域富集丰度增加，且 OSKMT 具有明显的高表达 MCL1 特征。通过 MCL1 的过表达与抑制，发现 MCL1 抑制影响了 OSKMT 的滋养层形成能力，从而确认了 MCL1 是 TBX3-MLL1 调控轴的关键下游基因。综上所述，本研究表明猪 TBX3 具有促进猪 iPSCs 向TLSCs的转变的功能，为发育生物学、疾病模型和家畜育种研究提供了新的平台。

Abstract: Pluripotent stem cells (PSCs) are useful for developmental and translational research because they have the potential to differentiate into all cell types of an adult individual. Pigs are one of the most important domestic ungulates, commonly used for food and as bioreactors. Generating stable pluripotent porcine PSC lines remains challenging. So far, the pluripotency gene network of porcine PSCs is poorly understood. Here we found that TBX3-derived induced pluripotent stem cells (iPSCs) closely resemble porcine 4-cell embryos with the capacity of totipotent-like stem cells (TLSCs). Interestingly, our data suggest that TBX3 facilitates the activation of H3K4me3 methyltransferase, specifically MLL1. Subsequent investigations revealed that the porcine 4-cell specific gene, MCL1, is a key downstream effector of the TBX3-MLL1 axis. Together, our study of the TBX3 regulatory network is helpful in the understanding of the totipotency characteristics of pigs.

Key words: TBX3 , porcine ,  induced pluripotent stem cells (iPSCs) ,  totipotency