In the first study by Zeng et al., the authors specifically isolated and sequenced ~7000 neoblasts with the goal of detecting a gene signature of a pluripotent neoblast, the potency of which was then functionally validated by gold-standard single-cell transplantations4. Based on sequencing, the authors detected 12 distinct neoblast subtypes in silico based on the similarity of gene expression. Importantly, the authors detected all previously identified neoblast subtypes. The authors then focused on a large subclass of neoblasts that they could not classify and found that this subtype, called Nb2, expressed high levels of a cell surface protein homolog of tetraspanin (tspan-1). Importantly, the authors made an antibody to TSPAN-1 and could, for the first time, prospectively isolate a TSPAN-1+ neoblast subpopulation by flow cytometry. To test the potency of TSPAN-1+ neoblasts, single cells were transplanted into hosts devoid of stem cells to test multilineage potential. The authors conclusively demonstrated that some of the TSPAN-1+ stem cells could restore the stem cell compartment, and thus, were functionally pluripotent.
While the Zeng et al., study found a method and gene signature to enrich pluripotent stem cells, there are some other interesting observations. First, while TSPAN-1- cells could not rescue the stem cell compartment, the TSPAN-1+piwi-1+ stem cells could only rescue the stem cell compartment of ~25% of animals following transplant. In the 2011 study, the rescue efficiency of single-cell transplants isolated only by morphology and without a cell surface marker was ~5%, so this study was a marked improvement in enriching for pluripotent neoblasts (or simply enriching for piwi-1+ cells). However, it remains unknown whether the 25% rescue in the Zeng study reflects the difficulty of the method (i.e., accidental killing of the transplanted stem cell), or whether this reflects true biological differences in potency. If it accurately reflects biology and only 25% of TSPAN-1+ stem cells are pluripotent, then there is much more room to hone in on the exact pluripotent stem cell population. Second, the Zeng study did not find a molecule that functioned specifically to maintain the TSPAN-1 population. Removal of TSPAN-1 function did not show loss of stem cells in a homeostatic context, and thus it remains unknown whether removal of TSPAN-1+ neoblasts would also remove pluripotency. Finally, the authors found that 89% of TSPAN-1+ cells were also piwi-1+, showing a high correlation of TSPAN-1 protein with the stem cell compartment, although the transcript for tspan-1 itself was difficult to detect athomeostasis. In the end, the Zeng model is attractive because the authors found TSPAN-1+ stem cells distributed throughout the stem cell compartment. Thus, virtually any injury fragment would inherit a pluripotent neoblast to restore any missing cell types (Fig.2a).
a Zeng et al., suggest that the Nb2 in silico cluster of neoblasts is the pluripotent population in planarians, which can be prospectively purified by TSPAN-1 protein expression and transplanted into hosts devoid of stem cells. Nb2 cells are distributed throughout the body axis (black dots) and specialized subtypes of neoblasts (various colored dots) are made from them in a traditional hierarchy. b Raz et al., show that although neoblasts can express factors that make them appear specialized (pink dot; FSTF+), they in fact can give rise to pluripotent-looking neoblasts (black dots; FSTF), which then make different lineages as well (blue). In this case, a neoblast may appear specialized (pink dot), but it can readily switch back to pluripotent state (black dots). It is a combination of cell cycle stage and down-regulating piwi-1 transcript the authors propose leads to true lineage commitment.
The second study, performed by Raz et al., stratified the >12,000 piwi-1+cells previously sequenced into the cell cycle stage based on gene expression5,6. Further, they sequenced several thousand new piwi-1+ cells taken from the 2C flow cytometry gate (representing G1/G0 stem cells) and the 4C gate (representing G2/M stem cells). The authors then examined the expression of known fate-specifying transcription factors (FSTFs) and observed an increase in FSTF expression as stem cells proceeded through the cell cycle. The authors showed that the 2 cells produced by a division often have an asymmetric expression of an FSTF in the two daughter cells: one that remains piwi-1[hi] and FSTF and the other that is piwi-1[low]FSTF+. Through careful analyses, the authors show, surprisingly, that FSTF+ stem cells can give rise to FSTF stem cells, implying that fate specification may either be reversible or simply adopted by a daughter cell at G2/M and that many or most piwi-1[hi] stem cells are pluripotent (Fig.2b). The Raz model is attractive because pluripotency can be accessed by most stem cells, and thus, these would be present in any given amputation fragment.
Interestingly, Raz et al. find that tspan-1+ (assayed using the additional co-expressed transcript tgs-1) stem cells largely express FSTFs toward neural fates and are not simply an FSTF, pluripotent cell state as was suggested by Zeng et al. However, it should be noted that the Raz et al., study was based on RNA expression (and investigating tgs-1 as a proxy for TSPAN-1). In contrast, Zeng et al. used prospective neoblast isolation based on protein expression investigating TSPAN-1. Thus, while the studies seem at odds, it remains possible that both are correct and that tgs-1+ stem cells are a mix of neural-specified and pluripotent. This could also explain the relatively low rescue percentage by a single-TSPAN-1+ neoblast in transplants at 25%.
Read more:
Finding the potency in planarians | Communications Biology - Nature.com
