Enrichment of Mouse Pituitary Thyrotropes from Hypothyroid Mice Using CD90-Based Fluorescence-Activated Cell Sorting

Thyrotropes are specialized endocrine cells in the anterior pituitary gland that secrete thyroid-stimulating hormone (TSH) and regulate thyroid activity. TSH secretion is positively regulated by hypothalamic thyrotropin-releasing hormone and negatively regulated by circulating thyroid hormones. Thyrotropes constitute only a small fraction of pituitary cells (~5%), complicating efforts to isolate and study their physiology. Here, we describe an enhanced approach for isolating thyrotropes from hypothyroid adult mice. Hypothyroidism was induced to expand the thyrotrope population prior to enzymatic dissociation of the anterior pituitary gland and fluorescence-activated cell sorting using the CD90.2 surface antigen. Processing anterior pituitary glands from twelve mice yielded approximately 1.5 × 10⁶ CD90.2-positive cells enriched for Tshb messenger RNA expression. CD90.2-positive cells represented approximately 25% of anterior pituitary cells and exhibited approximately fivefold higher Tshb expression compared with CD90.2-negative cells, indicating substantial thyrotrope enrichment. Isolated cells remained viable in short-term culture and retained responsiveness to triiodothyronine treatment. This protocol enables enrichment and culture of mouse thyrotropes for downstream molecular, transcriptomic, epigenomic, and physiological analyses.