Clonal Analysis of Planarian Stem Cells by Subtotal Irradiation and Single-Cell Transplantation

Stem cells, which both self-renew and produce differentiated progeny, represent fundamental biological units for the development, growth, maintenance, and regeneration of adult tissues. Characterization of stem cell lineage potential can be accomplished with clonal assays that interrogate stem cell output at the single-cell level. Here we present two methods for clonal analysis of individual proliferative cells (neoblasts) in the planarian Schmidtea mediterranea. The first method utilizes "subtotal" gamma irradiation to study rare surviving neoblasts and their clonal descendants in their native environment. The second method utilizes a fluorescent-activated cell sorting (FACS) strategy to obtain neoblast-enriched cell fractions, followed by single-cell transplantation into lethally irradiated hosts. Together, these methods provide a framework for generation and analysis of stem cell-derived clones in planarians.