Specific labeling of stem cell activity in human colorectal organoidsusing an ASCL2-responsive minigene

Organoid technology provides the possibility to culture human colontissue and patient-derived colorectal cancers (CRC) while maintainingall functional and phenotypic characteristics. Labeling of human colonstem cells (CoSCs), especially in normal and benign tumor organoids, ischallenging and therefore limits usability of multi-patient organoidlibraries for CoSC research. Here, we developed STAR (STem cell Ascl2Reporter), a minimal enhancer/promoter element that reportstranscriptional activity of ASCL2, a master regulator of LGR5+ CoSCfate. Among others via lentiviral infection, STAR minigene labels stemcells in normal as well as in multiple engineered and patient-derivedCRC organoids of different stage and genetic make-up. STAR revealed thatstem cell driven differentiation hierarchies and the capacity of cellfate plasticity (de-differentiation) are present at all stages of humanCRC development. The flexible and user-friendly nature of STARapplications in combination with organoid technology will facilitatebasic research on human adult stem cell biology.