Retaining pluripotency and exogenous mRNA introduction in planarian stem cell culture

Abstract

Planarians possess naturally occurring pluripotent adult somatic stem cells (neoblasts) required for homeostasis and whole-body regeneration. However, no reliable neoblast culture methods are currently available, hindering mechanistic studies of pluripotency and the development of transgenic tools. We report the first robust methods for neoblast culture and delivery of exogenous mRNAs. We identify optimal culture media for maintaining neoblasts in vitro, and show via transplantation that cultured stem cells retain pluripotency for two days. We developed a new procedure that significantly improves neoblast yield and purity by modifying standard flow cytometry methods. These methods enable the introduction and expression of exogenous mRNAs in neoblasts, overcoming a key hurdle impeding the application of transgenics in planarians. The advances in cell culture reported here create new opportunities for mechanistic studies of planarian adult stem cell pluripotency, and provide a systematic framework to develop cell culture techniques in other emerging research organisms.