Canonical and early lineage-specific stem cell types identified in planarian SirNeoblasts

The pluripotent stem cells in planarians, a model for tissue and cellular regeneration, have yet to be definitively identified. We recently developed a method to enrich piwi-1+ cells in Schmidtea mediterranea, by staining cells with SiR-DNA and Cell Tracker Green, named SirNeoblasts that permits their propagation and subsequent functional study in vivo. Since traditional enrichment for planarian neoblasts by Hoechst 33342 staining, to generate X1 cells, blocks the cell cycle, inducing cytotoxicity, this method represents a substantive technological advance for functional investigation of cell fate and regeneration in basic and applied research questions. However, the similarities in heterogeneity of cell subtypes between SirNeoblasts and X1 remain unknown. In this work, we performed single cell RNA sequencing for SirNeoblasts for comparison with differential expression patterns in a publicly available X1 single cell RNA sequencing data. We found first that all of the lineage-specific progenitor cells in X1 were present in comparable proportions in SirNeoblasts. In addition, SirNeoblasts contain an early muscle progenitor unreported in X1. Analysis of new markers for putative pluripotent stem cells identified here, with subsequent sub-clustering analysis revealed earlier lineages of epidermal, muscular, intestinal, and pharyngeal progenitors than have been observed in X1. Using the ski-3 and GCM markers, we also identified a pluripotent cell population at higher resolution than that provided by tgs-1. The use of SirNeoblast will enable broad experimental advances in regeneration and cell fate specification, given the possibility for propagation and transplantation of recombinant and mutagenized pluripotent stem cells not previously afforded to this rapid and versatile model system.