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

Summary

This protocol describes the enrichment of thyrotropes from hypothyroid adult mouse pituitary glands using enzymatic dissociation and CD90-based fluorescence-activated cell sorting, enabling downstream molecular analyses and short-term primary culture of a thyrotrope-enriched cell population.

Abstract

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.

Introduction

The pituitary gland is an endocrine organ located at the base of the brain that exerts hierarchical control over hormonal homeostasis. Its activity is regulated by hypothalamic signals that stimulate or inhibit the production of pituitary hormones, which in turn govern the function of peripheral endocrine organs. The gland contains a heterogeneous and dynamic population of cells, including stem/progenitor cells and differentiated hormone-producing cells¹. In response to physiological stress or injury, pituitary stem/progenitor cells can activate, self-renew, and differentiate into multiple hormone-secreting lineages²

Protocol

This protocol describes the isolation of thyrotropes from the AL of freshly dissected pituitary glands obtained from adult mice following at least 1 week of hypothyroidism induction. All animal procedures were performed in accordance with institutional and National Institutes of Health guidelines for the care and use of laboratory animals. The Institutional Animal Care and Use Committee of the University of Texas Medical Branch approved all animal procedures.

Male and female mice were pooled during the experiments because no sex-dependent differences in thyrotrope yield or downstream analyses were observed. Approximately 12 mice were used p....

Results

Twelve 8-week-old C57BL/6 mice (6 males and 6 females) were treated with MMI and KClO₄ in drinking water together with an iodine-deficient diet for 1 week to induce hypothyroidism (Figure 1A). Hepatic type 1 Dio1 mRNA expression was quantified in euthyroid and hypothyroid mice to confirm successful hypothyroidism induction²⁹. Hypothyroid mice exhibited an approximately 60% reduction in hepatic Dio1 mRNA expression compared with euthyroid contro.......

Discussion

The CD90-based FACS protocol described here provides a reproducible method for enriching primary thyrotropes from adult mouse pituitary tissue while preserving hormonal responsiveness. This method addresses a long-standing technical limitation in the study of thyrotrope biology and HPT axis regulation. Historically, investigations of thyrotrope regulation have relied heavily on the TαT1 cell line; however, TαT1 cells exhibit very low basal Tshb expression and do not consistently demonstrate canonical T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm filter	Corning	430769	For collection medium sterilization
0.22 µm syringe filter	Pall Life Sciences	PN4602	For collagenase sterilization
0.5% Trypsin–EDTA	Gibco	15400-054	For cell dissociation
1.5 mL low-binding microcentrifuge tubes	Invitrogen	AM12450	For FACS staining
15 mL polypropylene tubes	Falcon	352097	For anterior lobe collection and cell dissociation
23-gauge needle	BD Vacutainer Safety-Lok	367283	For cardiac perfusion
8-well glass chamber slide	Millipore	PEZGS0816	For post-FACS culture
Accudrop beads	BD Biosciences	345249	For drop delay calibration during FACS
Anti-mouse PE-Cy7 anti-CD90.2 antibody	Invitrogen	25-0902-82	For FACS staining
Automated cell counter	DeNovix	s-10700	For cell counting
Charcoal-stripped fetal bovine serum	Gibco	12676029	For post-FACS thyrotrope culture
Collagenase from Clostridium histolyticum	Sigma-Aldrich	C9263	For cell dissociation
Collection pan	Standard laboratory dissection tray	N/A	For cardiac perfusion
Custom-prepared HEPES-buffered saline solution (HBSS) without Ca2+ and Mg2+	N/A	N/A	For perfusion and cell dissociation
Cyclophilin A (CyA) oligonucleotide primer forward	Integrated DNA Technologies	5′-GCC GAT GAC GAG CCC TTG-3′	For RT-qPCR
Cyclophilin A (CyA) oligonucleotide primer reverse	Integrated DNA Technologies	5′-TGC CGC CAG TCG CAT TAT-3′	For RT-qPCR
DMEM, 1×	Gibco	11965-092	For collection medium
DNase I solution	Promega	M6101	For cell dissociation
Enoxaparin sodium injection	Sandoz	0781-3119-64	For perfusion
Fetal bovine serum (FBS)	Cytiva HyClone	SH30088.02HI	For collection medium
Flow cytometry staining buffer	Invitrogen	00-4222-26	For FACS staining
Hemostat	Fine Science Tools	13008-12	For thoracic cavity stabilization
HEPES, 1 M	Gibco	15630-080	For collection medium
HEPES, 1 M	Teknova	H1035	For HBSS preparation
Iodine-deficient diet	Envigo	TD.95007	For in vivo hypothyroidism induction
Iris forceps	Fine Science Tools	11065-07	For pituitary dissection
Isoflurane	Piramal Critical Care	66794-017-25	For anesthesia
L-glutamine	Gibco	35050-061	For collection medium
Medium 199	Gibco	11150-059	For post-FACS culture
Methimazole (MMI)	Sigma-Aldrich	M8506-100G	For in vivo hypothyroidism induction
MicroAmp Fast 96-well reaction plate	Applied Biosystems	4346907	For RT-qPCR
Penicillin–Streptomycin	Gibco	15140122	For post-FACS culture
Peristaltic pump	Fisher Scientific	GP1000	For cardiac perfusion
Potassium perchlorate (KClO4)	Sigma-Aldrich	241830-500G	For in vivo hypothyroidism induction
PowerTrack SYBR Green Master Mix	Applied Biosystems	A46109	For RT-qPCR
Propidium iodide	eBioscience	00-6990-50	For viability staining during FACS
QIAzol lysis reagent	Qiagen	79306	For RNA extraction
Silicone tubing	Cole-Parmer	EW-96410-13	For cardiac perfusion
Stereomicroscope	Nikon	SMZ-745T	For anterior lobe dissection
StepOnePlus Real-Time PCR System	Applied Biosystems	4376600	For RT-qPCR
Surgical spoon	Fine Science Tools	10090-13	For brain lifting during pituitary exposure
Transcriptor First Strand cDNA Synthesis Kit	Roche	4379012001	For cDNA preparation
Trypan Blue	Thermo Fisher Scientific	T10282	For cell counting
Tshb oligonucleotide primer forward	Integrated DNA Technologies	5′-TCT GTG CTG GGT ATT GTA TGA C-3′	For RT-qPCR
Tshb oligonucleotide primer reverse	Integrated DNA Technologies	5′-GCG GCT TGG TCG AGT AGT TG-3′	For RT-qPCR
Water bath	Fisher Scientific Isotemp Water Bath	15-462-5Q	For enzymatic digestion