Stem cells are at the heart of homeostasis, repair, disease, and cancer. Stem cell biology has evolved into a multifaceted field of research, captivating interest for both fundamental exploration and applications in regenerative medicine. This realm also encompasses physiological challenges such as aging. 

Adult stem cells occupy a central position in addressing many of the challenges encountered in fundamental research. They actively contribute to the homeostasis, regeneration, and repair of their respective tissues, exhibiting remarkable proliferative abilities and potential for cell fate determination. A comprehensive understanding of the signaling pathways and genetic or epigenetic modifications governing stem cells is crucial in this field. Functional assays are indispensable to attain a profound insight into these cells. 

In this collection, we focus on methods incorporating niche cues, including biophysical properties such as matrix stiffness and diffusible factors produced by niche cells, which significantly modulate stem cell function. When translating assays to in vitro settings, it is imperative to consider the three-dimensional nature of the stem cell environment. Integrating biophysical properties introduces the complexity of mechanical forces in regulating adult stem cell signaling. 

In vivo, meticulous control of various factors such as growth factors, morphogens, an appropriate immune environment, and extracellular matrix stiffness is essential. Additionally, the integration of in silico simulation for modeling stem cell behavior adds another dimension to our understanding. 

In summary, all methods, ranging from mouse models to organoids and primary cell cultures, that aid in assaying stem cell function deserve widespread recognition and an opportunity to be highlighted by the research community.