Tracing Stem and Progenitor Cells Using Sequential Administration of Three Thymidine Analogs In Vivo

Imaging of adult stem cells and their progeny in the gastrointestinal (GI) epithelium is useful for constructing cellular hierarchies, revealing stem cell location, and understanding stem cell dynamics, all of which provide an understanding of how they sustain tissues during homeostasis and injury. Pulse-chase approaches can be used to track stem and progenitor cells and understand tissue kinetics by using nucleotide analogs that irreversibly incorporate into DNA during S-phase. Existing pulse-chase approaches that use multiple analogs require harsh DNA denaturation which obscures nuclear staining and the ability to visualize colocalization of analog signals with DNA. This can prevent the ability to distinguish between diluted thymidine analog signal and auto fluorescent secretory granules in cells abundant in the GI epithelium. Here we build upon a method which combines three thymidine analogs for triple pulse-chase labeling of proliferating cells in mice to understand tissue kinetics and S-phase length while maintaining strong nuclear signals in the colon epithelium. This approach may be applied to other tissues to study cell proliferation kinetics.