Nematology Unit, Department of Biology, Ghent University, Ghent, Belgium.
PLoS One. 2012;7(1):e30227. doi: 10.1371/journal.pone.0030227. Epub 2012 Jan 19.
Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts) are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized 'Label-Retaining Cells' (LRCs). Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells. We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU- double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.
成体干细胞被认为获得了特殊的特征，以防止 DNA 复制错误的积累。有两种这样的机制，经常被认为是达到这个目的的机制，即细胞静止和 DNA 链在干细胞分裂过程中的非随机分离，这一理论被指定为不朽链假说。迄今为止，在干细胞系统中检验这两种机制的体内相关性一直很困难。已经表明，在扁形动物 Macrostomum lignano 多能干细胞（成体干细胞）存在于成年动物中。我们试图通过研究其分裂过程中 DNA 分离的确切模式，来解决 Macrostomum lignano 成体干细胞如何防止基因组错误积累的问题。在这项研究中，我们通过体内实验证明了细胞静止的四条证据。首先，通过 BrdU 脉冲追踪实验，我们定位了“标记保留细胞”（LRCs）。其次，EDU 脉冲追踪结合 Vasa 标记表明 LRCs 中存在成体干细胞，而大多数 LRCs 是分化细胞。我们表明，干细胞以缓慢的速度失去标记，表明细胞静止。第三，CldU/IdU- 双标记研究证实，保留标记的干细胞表现出低增殖活性。最后，肌动蛋白抑制剂细胞松弛素 D 的使用明确证明了 LRCs 中 DNA 链的随机分离。总之，我们的数据明确表明，Macrostomum lignano 中的大多数成体干细胞在细胞分裂过程中随机分配其 DNA，并且标记保留是细胞静止的直接结果，而不是标记链共分离的标志。
