Background

Fluorescent dyes that stain the nucleus in live cells are used for fluorescence-activated cell sorting (FACS) proliferating stem cells from planaria. In a seminal work, Hayashi et al. used Hoechst for staining planarian cells and categorized the cells into three populations, termed as X1, X2, and Xins population (Figure 2) and (Hayashi et al., 2006). This allowed sorting of the proliferating cells (X1 cells) in the planarian field of research (Hayashi et al., 2006). Later, Hoechst stain was found to be toxic so, for downstream applications where viable cells are required, a different FACS gate X1(fs) based on forward scatter (FSC) had to be used (Wagner et al., 2011). This population was obtained by back-gating the X1 population in an FSC vs SSC plot (Wang et al., 2018). However, X1(fs) is heavily contaminated by differentiated cells. Subsequently, the nuclear dye SiR-DNA was proposed as an alternative to Hoechst for obtaining proliferating stem cells with high viability (Lei et al., 2019; Niu et al., 2021). However, this flow cytometry method using a nuclear dye could not distinguish between the pluripotent and the specialized stem cell populations.

Methods that could distinguish pluripotent from specialized stem cells could advance our understanding of planarian stem cell biology and function. Since neoblasts have scant cytoplasm and reduced organelles, we reasoned that organelle complexity could be way to distinguish different cellular states (Higuchi et al., 2007). For this purpose, we used mitochondria, an organelle that plays an important role in metabolism and signaling. Studies in mammalian stem cells have suggested that the mitochondrial state, including the mitochondrial activity, content, morphology, and number varies between different cellular states. We have recently reported the use of MitoTracker Green (MTG) staining in combination with nuclear dyes, such as Hoechst and SiR-DNA, for the purification of pluripotent stem cells (Mohamed Haroon et al., 2021). Our data suggest that the population of proliferating cells with low MTG is enriched in pluripotent cells compared to the high MTG cells, as indicated by higher transplantation efficiency (~65% for MTG Low cells vs ~30% for MTG High cells). Further, this method could be used to enrich the 2N stem cells from the X2 population, which is a mixture of stem cells and mitotic progenitors. To this end, the X2 gate was divided into four populations based on MTG and FSC, which indicates size. We found that the 2N PSCs are enriched in the low MTG and high FSC gate (Mohamed Haroon et al., 2021). In contrast, X2 cells with low MTG and low FSC were predominantly early post-mitotic progenitors. The X2 high MTG cells, regardless of their size, were observed to be late progenitors (Mohamed Haroon et al., 2021). Here, we provide a step-by-step protocol for planarian dissociation, staining, and FACS gating of various MTG populations.