Ilastik: a machine learning image analysis platform to interrogate stem cell fate decisions across multiple vertebrate species.

Stem cells are the key cellular source for regenerating tissues and organs in vertebrate species. Historically, the investigation of stem cell fate decisions has been assessed in tissue sections using immunohistochemistry (IHC), where a trained user quantifies fluorescent signal in multiple randomly selected images using manual counting-which is prone to inaccuracies, bias, and is very labor intensive. Here, we highlight the performance of a recently developed machine-learning (ML)-based image analysis program called Ilastik using skeletal muscle as a model system. Interestingly, we demonstrate that Ilastik accurately quantifies Paired Box Protein 7 (PAX7)-positive muscle stem cells (MuSCs) before and during the regenerative process in whole muscle sections from mice, humans, axolotl salamanders, and short-lived African turquoise killifish, to a precision that exceeds human capabilities and in a fraction of the time. Overall, Ilastik is a free user-friendly ML-based program that will expedite the analysis of stained tissue sections in vertebrate animals.