Planaria are flatworms that can regenerate their entire body from a tiny tissue fragment. This remarkable property is mediated by neoblasts, highly heterogeneous populations of proliferating cells. Neoblasts are unusual because they include adult pluripotent stem cells (PSCs) — most organisms do not maintain PSCs beyond embryogenesis. However, characterization of these adult PSCs has been hampered by the inability to discriminate them from other neoblast subtypes. Now, a study in Cell reports the prospective identification and isolation of a neoblast subpopulation containing adult PSCs, the characterization of which should help determine the mechanisms underlying pluripotency and tissue regeneration.
Previous studies isolated a neoblast subpopulation enriched in PSCs (termed X1) from dissociated animal cells by fluorescence-activated cell sorting (FACS), but it contained only a small proportion of pluripotent cells capable of fully rescuing sublethally irradiated animals, which were termed clonogenic neoblasts (cNeoblasts). Other studies identified three classes of neoblasts (σ, ζ and γ) based on basic gene expression and functional profiles. The σ-class display some stem cell-like behaviour but, because they are defined empirically, they cannot be prospectively identified and isolated to determine if they are homogenous populations of cNeoblasts.
Zeng et al. set out to overcome this problem by defining prospective molecular signatures for different neoblast subpopulations. Neoblasts are known to express piwi-1, but the authors observed that levels of piwi-1 RNA varied between cells. Using FACS, they separated neoblasts according to their levels of piwi-1 RNA and PIWI-1 protein expression and showed that cells expressing the highest levels of both piwi-1 RNA and PIWI-1 protein (Piwi-1High) have characteristics of PSCs. However, similar to X1, only some Piwi-1High cells were pluripotent. Furthermore, they are transcriptionally distinct from X1 and σ-class cells, suggesting each is a different heterogeneous neoblast subpopulation containing cNeoblasts.
To identify molecular signatures representative of different subpopulations, the authors performed single-cell RNA sequencing (scRNA-seq) on 7,614 Piwi-1High X1 cells. The 7,088 cells that passed quality control clustered into 12 neoblast subpopulations (Nb1–Nb12), and discriminative gene sets were subsequently determined and validated for each cell cluster. The authors defined four criteria to determine which subclass most likely contained PSCs. First, the cell cluster should express high levels of piwi-1, σ-class gene markers and self-renewal regulator genes. Second, it should not express high levels of progenitor cell marker genes. Third, gene expression should increase after amputation to reflect the observed increase in PSC numbers in response to injury. Finally, it should have a distinct expression profile that reflects the ability of cNeoblast cells to survive sublethal irradiation. Only the Nb2 cluster — defined by expression of the tetraspanin-1 (TSPAN-1) membrane protein — met all four criteria, suggesting it alone contains pluripotent neoblasts. To confirm the stem cell properties of Nb2 cells, TSPAN-1-expressing cells were isolated by FACS for use in single-cell transplantation experiments; 23% of transplanted Nb2 cells rescued lethally irradiated animals — a 14-fold improvement over X1 rescue efficiency.
Subsequent transcriptional profiling experiments point to the huge potential afforded by the prospective identification and isolation of neoblast subpopulations. For instance, pluripotent Nb2 cells have been shown to undergo differential transcriptional responses to homeostasis, irradiation and injury.
Taken together, these studies suggest that in-depth characterization of PSCs and other neoblast subclasses throughout development is now a realistic prospect.
Zeng, A. et al. Prospectively isolated Tetraspanin+ neoblasts are adult pluripotent stem cells underlying planaria regeneration. Cell 173, 1593–1608 (2018)
About this article
Cite this article
Clyde, D. Prospecting for pluripotency. Nat Rev Genet 19, 471 (2018). https://doi.org/10.1038/s41576-018-0030-1