Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

Summary

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

Abstract

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Introduction

The pituitary is a tiny endocrine gland located at the base of the brain, where it is connected to the hypothalamus. The gland integrates peripheral and central (hypothalamic) inputs to generate a tuned and coordinated hormone release, thereby regulating downstream target endocrine organs (such as adrenal glands and gonads) for producing appropriate hormones at the proper time. The pituitary is the key regulator of the endocrine system and is therefore rightfully termed the master gland¹.

The mouse pituitary consists of three lobes (Figure 1), i.e., the anterior lob....

Protocol

Animal experiments for this study were approved by the KU Leuven Ethical Committee for Animal Experimentation (P153/2018). All mice were housed at the university's animal facility under standardized conditions (constant temperature of 23 ± 1.5 °C, relative humidity 40%-60%, and a day/night cycle of 12 h), with access to water and food ad libitum.

1. Mice

1. Use commercially available mouse strains, such as C57BL/6J mice, of young-adult age (8-12.......

Discussion

The AL-derived organoids, as described here, represent a powerful research model to study pituitary stem cells in vitro. At present, this organoid approach is the only available tool to reliably and robustly grow and expand primary pituitary stem cells. A pituitary organoid model derived from embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) has been reported previously, which closely recapitulates pituitary embryonic organogenesis²³; however, although highly useful to s.......

Materials

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Sigma-Aldrich	M6250
48-well plates, TC treated, individually wrapped	Costar	734-1607
A83-01	Sigma-Aldrich	SML0788
Advanced DMEM	Gibco	12491023
Albumin Bovine (cell culture grade)	Serva	47330
B-27 Supplement (50X), minus vitamin A	Gibco	12587010
Base moulds	VWR	720-1918
Buffer RLT	Qiagen	79216
Cassettes, Q Path Microtwin	VWR	720-2191
Cell strainer, 40 µm mesh, disposable	Falcon	352340
Cholera Toxin from Vibrio cholerae	Sigma-Aldrich	C8052
Deoxyribonuclease I from bovine pancreas	Sigma-Aldrich	D5025
D-glucose	Merck	108342
Dimethylsulfoxide (DMSO)	Sigma-Aldrich	D2650
DMEM, powder, high glucose	Gibco	52100039
Eppendorf Safe-Lock Tubes, 1.5 mL	Eppendorf	30120086
Epredia SuperFrost Plus Adhesion slides	Thermo Fisher Scientific	J1800AMNZ
Epredia HistoStar Embedding Workstation, 220 to 240Vac	Thermo Fisher Scientific	12587976
Ethanol Absolute 99.8+%	Thermo Fisher Scientific	10342652
Fetal bovine serum (FBS)	Sigma-Aldrich	F7524
GlutaMAX Supplement	Gibco	35050061
HEPES	Sigma-Aldrich	H4034
HEPES Buffer Solution	Gibco	15630056
InSolution Y-27632	Sigma-Aldrich	688001
L-Glutamine (200 mM)	Gibco	25030081
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-Free	Corning	15505739
Mr. Frosty Freezing Container	Thermo Fisher Scientific	5100-0001
N-2 Supplement (100X)	Thermo Fisher Scientific	17502048
N-Acetyl-L-cysteine	Sigma-Aldrich	A7250
Nunc Biobanking and Cell Culture Cryogenic Tubes	Thermo Fisher Scientific	375353
Paraformaldehyde for synthesis (PFA)	Merck	818715
PBS, pH 7.4	Gibco	10010023
Penicillin G sodium salt	Sigma-Aldrich	P3032
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122
Phenol red	Merck	107241
Potassium Chloride (KCl)	Merck	104936
Recombinant Human EGF Protein, CF	R&D systems	236-EG
Recombinant Human FGF basic/FGF2/bFGF (157 aa) Protein	R&D systems	234-FSE
Recombinant Human FGF-10	Peprotech	100-26
Recombinant Human IGF-1	Peprotech	100-11
Recombinant Human IL-6	Peprotech	200-06
Recombinant Human Noggin	Peprotech	120-10C
Recombinant Human R-Spondin-1	Peprotech	120-38
Recombinant Human/Murine FGF-8b	Peprotech	100-25
Recombinant Mouse Sonic Hedgehog/Shh (C25II) N-Terminus	R&D systems	464-SH
RNeasy micro kit	Qiagen	74004
SB202190	Sigma-Aldrich	S7067
SeaKem LE Agarose	Lonza	50004
Sodium Chloride (NaCl)	BDH	102415K
Sodium di-Hydrogen Phosphate 1-hydrate	PanReac-AppliChem	A1047
Sodium Hydrogen Carbonate (NaHCO3)	Merck	106329
Sodium-Pyruvate (C3H3NaO3)	Sigma-Aldrich	P5280
Stericup-GP, 0.22 µm	Millipore	SCGPU02RE
Steriflip-GP Sterile Centrifuge Tube Top Filter Unit, 0.22 μm	Millipore	SCGP00525
Sterile water	Fresenius	B230531
Streptomycin sulfate salt	Sigma-Aldrich	S6501
Syringe, with BD Microlance needle with intradermal bevel, 26G	BD Plastipak	BDAM303176
Thermo Scientific Excelsior ES Tissue Processor	Thermo Scientific	12505356
Titriplex III	Merck	108418
TrypL Express Enzyme (1X), phenol red	Thermo Fisher Scientific	12605028
Trypsin inhibitor from Glycine max (soybean)	Sigma-Aldrich	T9003
Trypsin solution 2.5 %	Thermo Fisher Scientific	15090046