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Background  The pluripotent stem cells in planarians, a model for tissue and cellular regeneration, remain further identification. 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 generates X1 cells, blocking the cell cycle and inducing cytotoxicity, this method by SiR-DNA and Cell Tracker Green represents a complementary technological advance for functional investigation of cell fate and regeneration. However, the similarities in heterogeneity of cell subtypes between SirNeoblasts and X1 remain unknown.    Results  In this work, we performed single cell RNA sequencing of 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 that is 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  gcm  as a marker, we also identified a cell subpopulation resided in previously identified  tgs-1 + neoblasts. Knockdown of  gcm  impaired the neoblast repopulation, suggesting a function of  gcm  in neoblasts.    Conclusions  In summary, the use of SirNeoblasts 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 that are not previously afforded to this rapid and versatile model system.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13619-021-00076-6.

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