Tsai Li-Kuang, Ou-Yang Huan, Xu Jie, Chen Chuan-Mu, Chang Wei-Fang, Sung Li-Ying
Institute of Biotechnology, National Taiwan University, Taipei, Taiwan.
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, Michigan, USA.
Stem Cells Dev. 2022 Nov;31(21-22):720-729. doi: 10.1089/scd.2022.0115. Epub 2022 Aug 22.
Haploinsufficiency of genes that participate in telomere elongation and maintenance processes, such as telomerase RNA component () and telomere reverse transcriptase (), often leads to premature aging-related diseases such as dyskeratosis congenita and aplastic anemia. Previously, we reported that when mouse tail tip fibroblasts (TTFs) were used as donor cells for somatic cell nuclear transfer (SCNT, also known as cloning), the derivative embryonic stem cells (ntESCs) had elongated telomeres. In the present work, we are interested to know if an additional round of SCNT, or recloning, could lead to further elongation of telomeres. TTFs were used to derive the first-generation (G1) ntESCs, followed by a second round of SCNT using G1- ntESCs as donor cells to derive G2- ntESCs. Multiple lines of G1- and G2- ntESCs were efficiently established, and all expressed major pluripotent markers and supported efficient chondrocyte differentiation in vitro. Compared with donor TTFs, telomere lengths of G1 ntESCs were elongated to the level comparable with that in wild-type ntESCs. Interestingly, recloning did not further elongate the telomere lengths of ntESCs. Together, our work demonstrates that while a single round of SCNT is a viable means to reprogram haploinsufficient cells to the ESC state, and to elongate these cells' telomere lengths, a second round of SCNT does not necessarily further elongate the telomeres.
参与端粒延长和维持过程的基因单倍剂量不足，如端粒酶RNA组分（）和端粒逆转录酶（），常常导致早衰相关疾病，如先天性角化不良和再生障碍性贫血。此前，我们报道当小鼠尾尖成纤维细胞（TTFs）用作体细胞核移植（SCNT，也称为克隆）的供体细胞时，衍生的胚胎干细胞（ntESCs）具有延长的端粒。在本研究中，我们想了解额外一轮的SCNT，即再克隆，是否会导致端粒进一步延长。使用TTFs衍生第一代（G1）ntESCs，随后以G1-ntESCs作为供体细胞进行第二轮SCNT以衍生G2-ntESCs。高效建立了多株G1-和G2-ntESCs，它们均表达主要的多能性标志物，并支持体外高效软骨细胞分化。与供体TTFs相比，G1 ntESCs的端粒长度延长至与野生型ntESCs相当水平。有趣的是，再克隆并未进一步延长ntESCs的端粒长度。总之，我们的研究表明，虽然一轮SCNT是将单倍剂量不足细胞重编程为ESC状态并延长这些细胞端粒长度的可行方法，但第二轮SCNT不一定会进一步延长端粒。
