Single‐cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its “index sorting” function for stem cell research

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.

Section: Introductionmentioning

confidence: 99%

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

Moritz¹,

Stöckle²,

Ortmeier³

et al. 2012

“…These findings, obtained at the end of the seventies A B Salò et al, 2009;Abril et al, 2010;Blythe et al, 2010;Hayashi et al, 2006Hayashi et al, , 2010Shibata et al, 2010;Aboobaker, 2011;Adamidi et al, 2011;Fernández-Taboada et al, 2011;Gentile et al, 2011;Qin et al, 2011). European planarian labs recently joined together by creating EuroPlanNet (www.europlannet.org), which brings together scientists with cross-disciplinary planarian expertise.…”

Section: What Are Your Most Interesting Findings and What Improvementmentioning

confidence: 95%

The past and present of planarians - An interview with Vittorio Gremigni

Salvetti¹,

Rossi²

2012

Vittorio Gremigni is a scientific leader in the field of planarian biology with a very long historical perspective. By using electron microscopy, he contributed to the reconstruction of the phylogenesis of free living "Turbellaria", and he pioneered the study of the origin of blastema cells by using chromosomal markers. In this interview, Professor Gremigni describes the steps that moved his career towards the planarian field, the main scientific achievements he obtained and the changes that are taking place in the field. He also discusses recent progress and unanswered questions on planarian neoblasts and regeneration.

“…To examine the relationship between DjP2X-A and cell proliferation, we analyzed the feeding-induced changes of two proliferative cell marker genes and DjP2X-A, by quantitative RT-PCR (qRT-PCR). We used Djpcna and DjMCM2 as proliferative cell marker genes, which are mainly expressed in the Sphase of the cell cycle in neoblasts (Hayashi et al, 2010). The expression level of Djpcna rapidly increased and peaked 12-24 hours after feeding, stayed above the steady state level until the third day after feeding, and reached a steady state level within 7 days (Fig.…”

Section: Djp2x-a Is Involved In Repressing Post-feeding Mitotic Burstsmentioning

confidence: 99%

The planarian P2X homolog in the regulation of asexual reproduction

Sakurai

Lee²,

Kashima³

et al. 2012

Self Cite

The growth in size of freshwater planarians in response to nutrient intake is limited by the eventual separation of tail and body fragments in a process called fission. The resulting tail fragment regenerates the entire body as an artificially amputated tail fragment would do, and the body fragment regenerates a tail, resulting in two whole planarians. This regenerative ability is supported by pluripotent somatic stem cells, called neoblasts, which are distributed throughout almost the entire body of the planarian. Neoblasts are the only planarian cells with the ability to continuously proliferate and give rise to all types of cells during regeneration, asexual reproduction, homeostasis, and growth. In order to investigate the molecular characteristics of neoblasts, we conducted an extensive search for neoblast-specific genes using the High Coverage Expression Profiling (HiCEP) method, and tested the function of the resulting candidates by RNAi. Disruption of the expression of one candidate gene, DjP2X-A (Dugesia japonica membrane protein P2X homologue), resulted in a unique phenotype. DjP2X-A RNAi leads to an increase of fission events upon feeding. We confirmed by immunohistochemistry that DjP2X-A is a membrane protein, and elucidated its role in regulating neoblast proliferation, thereby explaining its unique phenotype. We found that DjP2X-A decreases the burst of neoblast proliferation that normally occurs after feeding. We also found that DjP2X-A is required for normal proliferation in starved animals. We propose that DjP2X-A modulates stem cell proliferation in response to the nutritional condition.