Kato S et al. (2022), Effective enrichment of stem cells in regenerat...

XB-ART-59200

Dev Growth Differ 2022 Aug 01;646:290-296. doi: 10.1111/dgd.12797.

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Effective enrichment of stem cells in regenerating Xenopus laevis tadpole tails using the side population method.

Kato S , Kubo T , Fukazawa T .

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Xenopus laevis tadpoles 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 regenerating X. laevis tadpole 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 expressing pax7. The pax7-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 regenerating X. laevis tadpole tails, indicating that the combination of the SP method and scRNA-seq is useful for studying tissue stem cells that contribute to tail regeneration.

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Species referenced: Xenopus laevis
Genes referenced: foxc2 hes1 pax7

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Figure 1. Detection of SP fractions in X. laevis regeneration buds. (a) Schematic drawing of the SP cell preparation procedure from tadpole regeneration buds at 2 dpa. (b) FACS analysis of cells stained with Hoechst 33342 in the absence and presence of 200 μM verapamil. The fluorescence intensity of ~20,000 cells in two channels of 365–393 nm (Hoechst blue) and 650–670 nm (Hoechst red) is plotted. (c) Comparison of expression levels of marker genes estimated from RNA-seq data of SP-1 (magenta) and SP-2 (green).

Figure 2. Stem cell enrichment analysis using the SP method and scRNA-seq. (a) UMAP visualization of cells detected in the scRNA-seq data integrated with published scRNA-seq data of intact and regenerating tails at developmental stage 40 (Aztekin et al., 2019). Magenta dots indicate SP cells from tadpole regeneration buds at 2 dpa; gray dots indicate cells from intact and regenerating tails. Cell types were annotated based on the expression of known marker genes as described previously (Aztekin et al., 2019). (b) The proportion of SP cells in each cluster based on the cell numbers. (c) An extract of the clusters identified as muscle and the expression of marker genes of satellite cells (pax7.L), skeletal muscle (dupd1.S), and myotome (foxc2.S). (d) Dot plot showing expression of pax7.L, dupd1.S, and foxc2.S in muscle clusters of SP cells and intact and regenerating tails. Dot color indicates average expression; dot size represents the percentage of cells expressing each gene in the cluster. (e) An extract of neural clusters from UMAP and the expression of marker genes of neural progenitors (hes1.L) and differentiated neurons (tubb3.L). (f) An extract of muscle clusters of the SP cells from UMAP. Black arrowheads indicate cells that do not express pax7.L or differentiation markers of muscle lineage.