Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

Summary

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

Abstract

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5^{EGFP-IRES-CreERT2} allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

Introduction

In rodents, lingual taste buds are housed in fungiform papillae distributed anteriorly, bilateral foliate papillae posteriorly, as well as a single circumvallate papilla (CVP) at the posterodorsal midline of the tongue¹. Each taste bud is composed of 50-100 short-lived, rapidly renewing taste receptor cells (TRCs), which include type I glial-like support cells, type II cells that detect sweet, bitter, and umami, and type III cells that detect sour^2,3,4. In the mouse CVP, LGR5⁺ stem cells along the basal lamina produce all TRC types as well a....

Protocol

All the animal procedures were performed in an AAALAC-accredited facility in compliance with the Guide for the Care and Use of Laboratory Animals, Animal Welfare Act, and Public Health Service Policy, and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Anschutz Medical Campus. Lgr5^{EGFP-IRES-CreERT2} mice used in this protocol are from The Jackson Laboratory, Stock No. 008875.

NOTE: The following steps should be completed before beginning to ensure smooth and timely progression of the protocol: set water bath to 37 °C, set centrifuge to 4 °C, make injection and dissoc....

Results

Mice have one CVP, located posteriorly on the tongue, from which LGR5⁺ stem cells can be isolated (Figure 1A, black box). Injection of an enzyme solution under and around the CVP (Figure 1B) results in slight swelling of the epithelium and digestion of the connective tissue. Sufficient digestion is achieved following a 33 min incubation, which allows easy separation of the CVP epithelium from the underlying tissue. When attempting to .......

Discussion

Reported here is an efficient and readily repeatable method for culturing, maintaining, and processing lingual organoids derived from adult mouse taste stem cells. It was found that using three CVPs from 8 to 20-week-old Lgr5-EGFP mice is sufficient to obtain ~10,000 GFP⁺ cells for experimental use, resulting in 50 wells plated at a density of 200 cells per well in 48-well plates. Removal of CVP trench epithelia is optimized by injecting the lingual epithelium with freshly made Dispase II and type-I C.......

Materials

Name	Company	Catalog Number	Comments
Antibodies
Alexa Fluor 546 Donkey anti Goat IgG	Molecular Probes	A11056, RRID: AB_142628	1:2000
Alexa Fluor 546 Goat anti Rabbit IgG	Molecular Probes	A11010, RRID:AB_2534077	1:2000
Alexa Fluor 568 Goat anti Guinea pig IgG	Invitrogen	A11075, RRID:AB_2534119	1:2000
Alexa Fluor 647 Donkey anti Rabbit IgG	Molecular Probes	A31573, RRID:AB_2536183	1:2000
Alexa Fluor 647 Goat anti Rat IgG	Molecular Probes	A21247, RRID:AB_141778	1:2000
DAPI (for FACS)	Thermo Fischer	62247
DAPI (for immunohistochemistry)	Invitrogen	D3571, RRID:AB_2307445	1:10000
Goat anti-CAR4	R&D Systems	AF2414, RRID:AB_2070332	1:50
Guinea pig anti-KRT13	Acris Antibodies	BP5076, RRID:AB_979608	1:250
Rabbit anti-GUSTDUCIN	Santa Cruz Biotechnology Inc.	sc-395, RRID:AB_673678	1:250
Rabbit anti-NTPDASE2	CHUQ	mN2-36LI6, RRID:AB_2800455	1:300
Rat anti-KRT8	DSHB	TROMA-IS, RRID: AB_531826	1:100
Equipment
2D rocker	Benchmark Scientific Inc.	BR2000
3D Rotator	Lab-Line Instruments	4630
Big-Digit Timer/Stopwatch	Fisher Scientific	S407992
Centrifuge	Eppendorf	5415D
CO2 tank	Airgas	CD USP50
FormaTM Series 3 Water Jackeed CO2 Incubator	Thermo Scientific	4110	184 L, Polished Stainless Steel
Incucyte	Sartorius	Model: S3	Cancer Center Cell Technologies Shared Resource, University of Colorado Anschutz Medical Campus
MoFlo XDP100	Cytomation Inc	Model: S13211997	Gates Center Flow Cytometry Core, University of Colorado Anschutz Medical Campus
Orbital Shaker	New Brunswick Scientific	Excella E1
Real-Time PCR System	Applied Biosystems	4376600
Refrigerated Centrifuge	Eppendorf	5417R
Spectrophotometer	Thermo Scientific	ND-1000
Stereomicroscope	Zeiss	Stemi SV6
Thermal Cycler	Bio-Rad	580BR
Vortex	Fisher Scientific	12-812
Water bath	Precision	51220073
Media
A83 01	Sigma	SML0788-5MG	Stock concentration 10 mM, final concentration 500 nM
Advanced DMEM/F12	Gibco	12634-010
B27 Supplement	Gibco	17504044	Stock concentration 50X, final concentration 1X
Gentamicin	Gibco	15750-060	Stock concentration 1000X, final concentration 1X
Glutamax	Gibco	35050061	Stock concentration 100X, final concentration 1X
HEPES	Gibco	15630080	Stock concentration 100X, final concentration 1X
Murine EGF	Peprotech	315-09-1MG	Stock concentration 500 µg/mL, final concentration 50 ng/mL
Murine Noggin	Peprotech	250-38	Stock concentration 50 µg/mL, final concentration 25 ng/mL
N-acetyl-L-cysteine	Sigma	A9165	Stock concentration 0.5 M, final concentration 1 mM
Nicotinamide	Sigma	N0636-100g	Stock concentration 1 M, final concentration 1 mM
Pen/Strep	Gibco	15140-122	Stock concentration 100X, final concentration 1X
Primocin	InvivoGen	ant-pm-1	Stock concentration 500X, final concentration 1X
SB202190	R&D Systems	1264	Stock concentration 10 mM, final concentration 0.4 µM
WRN Conditioned media			Received from Dempsey Lab (AMC Organoid and Tissue Modeling Share Resource). Derived from L-WRN (ATCC® CRL-3276™) cells
Y27632 dihydochloride 10ug	APExBIO	A3008-10	Stock concentration 10 mM, final concentration 10 µM
Other
1 ml TB Syringe	BD Syringe	309659
2-Mercaptoethanol, min. 98%	Sigma	M3148-25ML	β-mercaptoethanol
2.0 mL Microcentrifuge Tubes	USA Scientific	1420-2700
48-well plates	Thermo Scientific	150687
5 3/4 inch Pasteur Pipets	Fisherbrand	12-678-8A
Albumin from bovine serum (BSA)	Sigma Life Science	A9647-100G
Buffer RLT Lysis buffer	QIAGEN	1015750
Cell Recovery Solution	Corning	354253
Cohan-Vannas Spring Scissors	Fine Science Tools	15000-02
Collagenase from Clostridium histolyticum, type I	Sigma Life Science	C0130-1G
Cultrex RGF BME, Type 2, Pathclear	R&D Systems	3533-005-02	Matrigel
Dispase II (neutral protease, grade II)	Sigma-Aldrich (Roche)	4942078001
Disposable Filters	Sysmex	04-0042-2316
Dulbecco’s Phosphate Buffered Saline pH 7.4 (1X) (Ca2+ & Mg2+ free)	Gibco	10010-023
Dulbecco’s Phosphate Buffered Saline with Ca2+ & Mg2+	Sigma Life Sciences	D8662-500ML
Dumont #5 Forceps	Fine Science Tools	11252-30
EDTA, 0.5M (pH 8.0)	Promega	V4231
Elastase Lyophilized	Worthington Biochemical	LS002292
Extra Fine Bonn Scissors	Fine Science Tools	14084-08
Fetal Bovine Serum (FBS)	Gibco	26140-079
Fluoromount G	SouthernBiotech	0100-01
HEPES Solution	Sigma Life Science	H3537-100ML
HyClone Tryspin 0.25% + EDTA	Thermo Scientific	25200-056
iScript cDNA Synthesis Kit	Bio-Rad	1706691
Modeling Clay, Gray	Sargent Art	22-4084
Needle	BD Syringe	305106
Normal Donkey Serum	Jackson ImmunoResearch	017-000-121
Normal Goat Serum	Jackson ImmunoResearch	005-000-121
Paraformaldehyde	Sigma-Aldrich	158127
PowerSYBR Green PCR Master Mix	Applied Biosystems	4367659
RNeasy Micro Kit	QIAGEN	74004
Safe-Lock Tubes 1.5 mL	Eppendorf	022363204
Sodium Chloride	Fisher Chemical	7647-14-5
Sodium Phosphate dibasic anhydrous	Fisher Chemical	7558-79-4
Sodium Phosphate monobasic anhydrous	Fisher Bioreagents	7558-80-7
SuperFrost Plus Microscope Slides	Fisher Scientific	12-550-15
Surgical Scissors - Sharp	Fine Science Tools	14002-14
Triton X-100	Sigma Life Science	T8787-100ML
VWR micro cover glass	VWR	48366067	22x22mm