Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Summary

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Abstract

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

Introduction

Mouse incisors grow continuously due to life-long preservation of the stem cells (SC) that support the unceasing production of tooth components. These include epithelial SCs, which generate enamel-producing ameloblasts, and mesenchymal stem cells (MSCs), which generate dentin-producing odontoblasts, among other cells¹. The epithelial SCs in the continuously growing incisors were initially identified as label-retaining cells^2,3 and have since been shown to express a number of well-known stemness genes, including Sox2⁴. These cells share common features with epithe....

Protocol

This protocol involves the use of animals and all the procedures were approved by Ethical Committees on the Use and Care of Animals and the Animal Facility at the University of Helsinki.

1. Preparation of the organ culture dish

1. Perform all procedures in a laminar flow hood. Clean work surfaces with 70% ethanol and use autoclaved glass instruments and solutions. Sterilize scissors and other metal equipment in a glass-bead sterilizer.
2. Prepare filters normally stored in 70% ethanol by washing them three times in 1x PBS to remove ethanol (Figure 1). Cut filters into rectangular pieces (3 x 3-5....

Results

The epithelial SCs reside in a niche called the cervical loop, which is located at the proximal end of the incisor (Figure 3A). Cervical loops are morphologically distinct structures composed of inner and outer enamel epithelium that encase the stellate reticulum, a core of loosely arranged epithelial cells (Figure 3B,C). There are two cervical loops in each incisor (Figure 3A), but only the labial cervical loop con.......

Discussion

In vitro organ culture has been used extensively to study inductive potential and epithelial-mesenchymal interactions that govern organ growth and morphogenesis. The Thesleff laboratory has demonstrated how to adapt the Saxén modification of the Trowell-type organ culture and use it to study tooth development¹⁴. The reproducible conditions and advancements in fluorescent reporters have made this a useful method for monitoring tooth morphogenesis and the distinct cell populations with.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-mL plastic syringes
Disposable 20/26 gauge hypodermic needles	Terumo
DMEM	Gibco	61965-026
Dulbecco's Phosphate-Buffered Saline	Gibco	14287
Extra Fine Bonn Scissors	F.S.T.	14084-08
F-12	Gibco	31765-027
FBS South American (CE)	LifeTechn.	10270106	divide in aliquotes, store at -20°C
Glass bead sterilizer, Steri 250 Seconds-Sterilizer	Simon Keller Ltd	4AJ-6285884
GlutaMAX-1 (200 mM L-alanyl-L-glutamine dipeptide)	Gibco	35050-038
Isopore Polycarb.Filters, 0,1 um 25-mm diameter	MerckMillipore	VCTP02500	Store in 70% ethanol at room temperature.
L-Ascobic Acid	Sigma	A4544-25g	diluted 100 mg/ml in MilliQ, filter strerilized and divided in 20μl aliquotes, store at dark, -20°C
Low melting agarose	TopVision	R0801
Metal grids			Commercially available, or self-made from stainless-steel mesh (corrosion resistant, size of mesh 0.7 mm). Cut approximately 30 mm diameter disk and bend the edges to give 3 mm height. Use nails to make holes.
Micro forceps	Medicon	07.60.03
Paraformaldehyde	Sigma-Aldrich
Penicillin-Streptomycin (10,000U/ml) sol.	Gibco	15140-148
Petri dishes, Soda-Lime glass	DWK Life Sciences	9170442
Petridish 35 mm, with vent	Duran	237554008
Petridish 90 mm, no vent classic	Thermo Fisher	101RT/C
Small scissors