An improved clonogenic culture method for thymic epithelial cells.

A clonogenic assay system for thymic epithelial cells (TECs) is of crucial importance for identifying thymic epithelial stem and/or progenitor cells, evaluating their activities, and understanding the mechanisms of thymic involution. However, current systems are not sufficiently sensitive at detecting and quantifying TEC colonies from the adult thymus. Here, we optimized the culture condition to detect visible colonies from adult TECs by modifying our previous culture methods. Epidermal growth factor and leukemia inhibitory factor significantly enhanced the colony-forming efficiency of total TECs from embryo as well as adult mice when added 3 days after plating. Importantly, characteristics of the TEC colonies formed by the improved condition were almost equivalent to those by the original culture condition with respect to self-renewal and the expression of cell surface markers and intracellular keratins. Furthermore, the colonies derived from total TECs showed immature phenotypes and generated both mature cortical TECs and medullary TECs upon implantation in vivo. These data indicate a more sensitive clonogenic assay system for TECs was established and suggest the improved culture condition supports the colony formation of stem/progenitor cells for cTECs, mTECs and/or bipotent TECs.