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 epithelial SCs in other organs and reside within the SC niche called the cervical loop located on the labial side of the incisor. The niche is a dynamic entity composed of cells and extracellular matrix that control SC activity⁵. Lineage-tracing studies have demonstrated that Sox2+ epithelial SCs can regenerate the whole epithelial compartment of the tooth and that they are crucial for successional tooth formation^6,7. MSCs with dentin reparative or regenerative potential are largely recruited from outside the organ through blood vessels and nerves^8,9,10,11, therefore, providing a suitable model to study recruitment, migration, and housing of the MSC population.

To study SCs in vivo is not always feasible, since many of the genetic and/or pharmacological manipulations can affect organ homeostasis and/or have lethal consequences. Therefore, organ culture provides an excellent tool to study regulation of SCs and their niches in vitro. The organ culture system that utilizes a metal grid was initially developed by Trowell¹² to study organ development and has been further modified by Saxen¹³ to study inductive signals in kidney development. Since then, this in vitro method of culturing the whole or part of the organ has been successfully applied in different fields. In the field of tooth development, this method has been widely used to study the epithelial-mesenchymal interactions that govern tooth development¹⁴ and successional tooth formation¹⁵. The work of the Thesleff laboratory has demonstrated the utility of this system for temporal analysis of tooth growth and morphogenesis, for analysis of the effect of various molecules and growth factors on tooth growth, and for time-lapse live imaging of tooth development^16,17. More recently, this method has been utilized to study regulation of incisor SCs and their niche^18,19, which is described in detail here.

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 x 5 mm).
   NOTE: Washed filter pieces can be stored for several days in 1x PBS at 4 °C.
3. Prepare the culture medium (1:1 DMEM:F12 supplemented with 1% [v/v] 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl, 10% [v/v] FBS, 150 µg/mL ascorbic acid, and 0.2% [v/v] penicillin [10,000 I.U./mL] and streptomycin [10,000 µg/mL]). Store at 4 °C.
4. Place the 30 mm metal grids (with 1-2 mm diameter holes that enable tissue imaging) in a 35 mm Petri dish. Add sufficient media to reach the grid surface without producing air bubbles. Pre-warm the prepared culture dish at 37 °C until the tissue is isolated and ready for culture (in a standard incubator with 5% CO₂ and 90%-95% humidity).

2. Incisor dissection and isolation of the proximal end

1. Sacrifice the animals following an approved animal care protocol.
2. Decapitate the mouse and dissect the mandible. To do so, first remove the skin to expose the mandible and cut through the masseter muscles to separate it from the maxilla and the rest of the head.
3. Once the mandible is isolated, remove the tongue and as much soft tissue as possible.
4. Collect all the mandibles and keep them in a Petri dish containing PBS on ice, as this enhances the viability of the tissues.
5. Transfer one mandible to a glass Petri dish and use disposable 20/26 G hypodermic needles to dissect the incisor under a stereomicroscope. Split the mandible at the midline, cutting through the symphysis. Clean the soft and muscle tissue away from the bone surface for better visualization.
   ​NOTE: A glass Petri dish is essential, as this will not blunt the needles.
6. Mandibles obtained from animals younger than 10 days are softer and more fragile, therefore, use disposable 20/26 G hypodermic needles to open each half of the mandible longitudinally to expose the incisor tooth. For mice older than 10 days, use tweezers to grip the mandible and break the bone to expose the tooth.
7. Gently detach the incisor from the surrounding bone and cut off the proximal end, which contains the cervical loop.
8. Cut the proximal end and remove the mineralized enamel and dentin matrix.
9. Keep the collected proximal ends in Dulbecco's PBS on ice until ready for culture.

3. Culture

1. Carefully place a filter rectangle on the top of a grid in a pre-warmed culture dish.
2. Use a stereomicroscope to properly orient the tissue pieces.
3. Place in a standard incubator at 37 °C with 5% CO₂ and 90%-95% humidity.
4. Change the medium every other day and replace with fresh medium, carefully avoiding formation of air bubbles. Monitor tissue growth and photograph daily using a camera attached to the stereomicroscope.

4. Adding soluble factors to the culture

1. Supplement the culture medium with soluble factors and molecules of interest to study their effect on regulation of SCs.
   ​NOTE: The administration protocol for any molecule (growth factors, signaling molecules, blocking antibodies, pharmacological compounds such as inhibitors or activators, vectors, etc.) depends on its half-life and solubility. These parameters also determine the appropriate control to be used.

5. Molecular and histological analyses

1. Remove the culture medium.
2. Carefully add ice-cold methanol to the tissues to prevent detachment from the filters.
3. Leave methanol for 5 min.
4. Transfer filters carrying tissue explants to sampling tubes.
5. Fix the explants in 4% paraformaldehyde in PBS for 10-24 h at 4 °C.
6. Proceed with established protocols for histological processing (paraffin, frozen, etc.) or immunostainings.

6. Culture of tissue slices

NOTE: There are several variations to this protocol, which are all equally successful and are a matter of personal choice, depending on the speed and the skill of the user. These refer to the buffer used to collect and maintain tissue viability. For this purpose, Krebs buffer, PBS supplemented with 2% glucose and antibiotics, or PBS can be used. If Krebs buffer is used, it should be made 1 day in advance and kept at 4 °C.

1. Before the experiment, prepare 4%-5% low-melting point agarose by dissolving 2-2.5 g of low-melting point agarose in 50 mL of boiling 2% glucose/PBS, after which the solution should be placed in a 45 °C water bath.
2. Set up the vibratome, wash with 70% ethanol, and fill with ice-cold 2% glucose/PBS supplemented with antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin).
3. Dissect the proximal ends of the incisor and collect them in the ice-cold 2% glucose/PBS supplemented with antibiotics.
4. Place one proximal end of the incisor in the mold containing 4%-5% low-melting point agarose. Under a stereomicroscope, orient the piece in the desired direction and leave on ice for the agarose to harden.
5. Trim the hardened agarose block and place it in the vibratome. Cut thick slices (150-300 µm).
6. Collect the slices in a Petri dish containing ice-cold 2% glucose/PBS supplemented with antibiotics.
7. Use a spatula to transfer the thick slices on a filter rectangle placed on the pre-warmed grid prepared as in section 1.3.
8. Incubate the thick slices in the incubator and proceed with imaging (Figure 2).

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

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