Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Summary

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Abstract

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest^1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal's life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

Introduction

One of the unique characteristics of vertebrates is the evolution of teeth. The tooth has become an important model system for many areas of research, as the molecular pathways and morphological specializations relevant to this organ have been investigated from several perspectives, including by developmental and evolutionary biologists⁵. More recently, the field of regenerative medicine has begun to gain valuable insights using the tooth as a model. In particular, the discovery of dental epithelial stem cells has been an important advance^6-13.

All rodents possess continuously growing incisors whose growth is fueled by....

Protocol

1. Dissect Lower Hemi-mandibles from Adult Mouse

1. Prior to performing this protocol, obtain necessary institutional approval and be sure to comply with all animal care guidelines. Euthanize animal using standard IACUC approved procedures. For this work CO₂ asphyxiation was used followed by cervical dislocation.
   1. OPTIONAL: Separate head from the rest of the body using a razor blade.
2. Remove skin from the lower lip to the neckline.
3. Using a scalpel, make an incision between the two lower incisors, which are loosely connected. Upon applying pressure, the mandible will be split into two halves.
4. Wed....

Results

The mouse hemi-mandible comprises one continuously growing incisor and three rooted molars (Figure 1A). All teeth consist of dentin and enamel, the two mineralized components of the tooth crown (Figures 1A and 1A'). The incisor houses two stem cell niches called the labial CL and lingual CL, and enamel is formed exclusively on the labial side (Figure 1A'). DESCs are responsible for incisor enamel formation and are housed in the labial CL, specifically th.......

Discussion

Epithelial cells were first successfully cultured over 40 years ago^21-24, and more recently, successful isolation of epithelial stem cells^25-27 has advanced our knowledge of epithelial biology. We report a protocol for isolating the DESCs of the adult mouse incisor, a relatively understudied population of stem cells that has the potential to yield important insights into dental biology and enamel formation. This protocol was initially based on previous reports of epithelial stem cell isolation from .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Forceps, size 5	Fine Science Tools	11251-30	FST by Dumont, Dumoxel
Forceps straight, fine, 3.5	Roboz	RS5070
Razorblades	Electron Microscopy Services	#71960
Scalpel Handle No. 3	VWR
Feather Blade No. 15	Electron Microscopy Services	#72044-15	Surgical Stainless Steel
Collagenase Type-1 filtered	Worthington Biochemical Corporation	#4214
Insulin syringe	BD	#329424
Accumax	EMD Millipore	#SCR006
DMEM/F12	Gibco	11320-033
EGF	R&D	2028-EG-200
FGF2	R&D	233-FB-025
B27 Supplement	Gibco	10889-038