Effective enrichment of stem cells in regenerating Xenopus laevis tadpole tails using the side population method
Effective enrichment of stem cells in regenerating Xenopus laevis tadpole tails using the side population method
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DOI：
10.1111/dgd.12797
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发表时间：
2022-07-31
影响因子：
2.5
通讯作者：
Fukazawa，Taro
中科院分区：
文献类型：
--
作者：
Kato，Sumika;Kubo，Takeo;Fukazawa，Taro
非洲爪哇具有很强的再生能力，在断尾后能再生出完整的尾巴。谱系受限的组织干细胞被认为是通过产生未分化的祖细胞来响应尾部截肢而为再生组织提供来源的。然而，由于组织干细胞的稀有性，很难阐明组织干细胞在尾巴再生过程中的行为动力学，而且在两栖动物中分离这些细胞的方法也很少。在这里，为了检测和分析稀有组织干细胞，我们试图从尾部再生芽中丰富组织干细胞。高Hoechst染料外排能力被认为是几种类型的哺乳动物组织干细胞的共同特征；这些干细胞被指定为“侧群(SP)”，可以通过流式细胞术(SP方法)进行浓缩。目的：评价SP法浓缩干细胞再生X细胞的效果。我们对再生芽中的SP细胞进行了单细胞RNA测序(scRNA-seq)，并分析了表达pax7的肌肉干/祖细胞中卫星细胞的频率。与正常尾巴和再生芽相比，表达pax7的细胞在SP中更为丰富。此外，被认为是神经干/祖细胞的HES1表达细胞也在SP中富含。我们的研究结果表明，SP法是成功富含再生X中的组织干细胞的有效方法。这表明SP方法和scRNA-seq相结合对于研究有助于尾巴再生的组织干细胞是有用的。
Xenopus laevistadpoles have a strong regenerative ability and can regenerate their whole tails after tail amputation. Lineage‐restricted tissue stem cells are thought to provide sources for the regenerating tissues by producing undifferentiated progenitor cells in response to tail amputation. However, elucidating the behavioral dynamics of tissue stem cells during tail regeneration is difficult because of their rarity, and there are few established methods of isolating these cells in amphibians. Here, to detect and analyze rare tissue stem cells, we attempted to enrich tissue stem cells from tail regeneration buds. High Hoechst dye efflux capacity is thought to be a common characteristic of several types of mammalian tissue stem cells; these stem cells, designated as the “side population (SP),” may be enriched by flow cytometry (SP method). To evaluate the effectiveness of stem cell enrichment using the SP method in regeneratingX. laevistadpole tails, we performed single‐cell RNA sequencing (scRNA‐seq) of SP cells from regeneration buds and analyzed the frequency of satellite cells, which are muscle stem/progenitor cells expressingpax7. Thepax7‐expressing cells were enriched in the SP compared with whole normal tails and regeneration buds. Furthermore,hes1‐expressing cells, which are assumed to be neural stem/progenitor cells, were also enriched in the SP. Our findings suggest that the SP method is efficient for successfully enriching tissue stem cells in regeneratingX. laevistadpole tails, indicating that the combination of the SP method and scRNA‐seq is useful for studying tissue stem cells that contribute to tail regeneration.