GSE107874
A tetraspanin family member functionally resolves and facilitates the purification of adult pluripotent stem cells used for whole-body regeneration (Bulk)
Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) capable of sustaining tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single cell transplantation to address this long-standing issue. Large-scale scRNA-seq of fluorescently sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein, and marked by a conserved cell-surface protein coding gene, tetraspanin 1 (tspan1). tspan1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody rescued with high efficiency the viability of lethally irradiated animals after single-cell transplantation. Our work demonstrates the first prospective isolation of an adult PSC, bridges a conceptual dichotomy between operationally and molecularly defined neoblasts, and sheds light on the mechanisms governing in vivo pluripotency and the source of regeneration in planarians. Overall design: 1) Examine gene expression profiles of FACS sorted X1, X2 and Xins cell populations from adult flatworms. The experiment was performed in triplicate, resulting in 9 samples; 2) Examine gene expression profiles of wildtype untreated and lethal (10K) irradiated animals. The experiment was performed in triplicate, resulting in 6 samples; 3) Examine gene expression profiles of Flow Cytometry sorted PIWI-1_high,PIWI-1_low and PIWI-1_negative cell populations from adult flatworms. The experiment was performed in quadruplicate, resulting in 12 samples; 4) To examine gene expression profiles of regenerating fragment over the regeneration time-course, we performed RNA-seq on 17 time-points of regeneration, starting from amputation of small-sized fragments to fully functional regenerated animals. The experiment was performed in quadruplicate, resulting in 68 samples.