Heterogeneity of planarian stem cells in the S/G2/M phase

Abstract

The planarian adult stem cell (pASC) population has a specific molecular signature and can be easily visualized and isolated by flow cytometry. However, the lack of antibodies against specific surface markers for planarian cells prevents a deeper analysis of specific cell populations. Here, if we describe the results of the immunoscreening of pASC plasma membrane proteins (PMPs). A novel papain-based method for planarian cell dissociation enabling both high yield and improved cell viability was used to generate single cell preparations for PMP purification. PMPs were used for intraperitoneal immunization of mice and thus about 1000 hybridoma clones were generated and screened. Supernatants collected from the hybridoma clones were first screened by ELISA and then by live immuno-staining. About half of these supernatants stained all the planarian cells, whereas the other half specifically labeled a subfraction thereof. A detailed analysis of two hybridoma supernatants revealed that large subfractions of the X1, X2 and Xin populations differentially express specific membrane markers. Quantitative PCR data disclosed a correlation between the immunostaining results and the expression of markers of the early and late progeny, also for those pASCs in the S/G2/M phase of the cell cycle (X1 population). Thus, about two thirds of the cycling pASCs showed a specific membrane signature coupled with the expression of markers hitherto considered to be restricted to differentiating, post-mitotic progeny. In summary, a library of 66 monoclonal antibodies against planarian PMPs was generated. The analysis of two of the clones generated revealed that a subset of cells of the X1 population expresses early and late progeny markers, which might indicate that these cells are committed while still proliferating. The findings demonstrate the usefulness of our PMP antibody library for planarian research.

JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

“…Expression analysis of h2b by FISH and qRT-PCR showed that the proportion of neoblasts in JNK(RNAi) animals was similar to that of controls (Figure S5C-S5D). In agreement, sorting of the different planarian cell populations by FACS, a method by which planarian cells can be separated based on their DNA content and size [35], [36], showed no alterations in the proportion of actively cycling cells (in the S or G2/M phase of the cell cycle; ×1 sub-population) after JNK RNAi (Figure S5E). Therefore, although JNK is expressed in neoblasts and essential for regeneration, it is not required for their viability.…”

“…The dissociation of planarians, cellular labeling, and isolation of cells by FACS were performed as described previously [36].…”

Single-Cell Analysis Reveals Functionally Distinct Classes within the Planarian Stem Cell Compartment

“…Recently, gene expression signatures specific to differentiated tissues were shown to mark small numbers of lineage-specified neoblasts during regeneration (Cowles et al, 2013; Currie and Pearson, 2013; Lapan and Reddien, 2011; Scimone et al, 2011) and in homeostasis (Hayashi et al, 2010; Moritz et al, 2012). In addition, neoblasts can be subdivided with respect to an otherwise uncharacterized surface antigen (Moritz et al, 2012) or a trans-splicing sequence (Rossi et al, 2014). However, as a consequence of small numbers and/or lack of functional characterization, it remains unknown whether the above phenomena reflect the presence of substantial cellular classes within the neoblast population.…”

The EGFR signaling pathway controls gut progenitor differentiation during planarian regeneration and homeostasis

“…Neoblasts are the only dividing cells of planarians (Morita and Best, 1984;Newmark and Sánchez Alvarado, 2000) and thus can differentiate into any cell type. Accordingly, the neoblast population consists of a compartment of pluripotent ASCs (Wagner et al, 2011) and a heterogeneous pool of lineage-restricted progenitors (Hayashi et al, 2010;Moritz et al, 2012;Reddien, 2013;Scimone et al, 2014;van Wolfswinkel et al, 2014), which can be distinguished by the expression of tissue-specific transcription factors (Adler et al, 2014;Cowles et al, 2013;Lapan and Reddien, 2012;Scimone et al, 2014Scimone et al, , 2011Wagner et al, 2011). Despite recent advances, the genes and molecular pathways that regulate the differentiation of known progenitors into distinct mature cell types remain poorly understood.…”