Name: Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor (Video) | JoVE Journal
Uploaded: 2014-05-01T13:00:00.000+00:00
Duration: 8 min 14 s
Description: Learn to isolate and culture dental epithelial stem cells from the adult mouse incisor using microdissection techniques. This reliable method enables consistent cell culture for dental tissue regeneration research.

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5 minutes

The authors thank Xiu-Ping Wang for initial help with DESC cultures. The authors are funded in part by fellowships and grants from the National Institutes of Health (K99-DE022059 to A.H.J., K12-GM081266 to M.G.C., K08-DE022377-02 to O.H., and R01-DE021420 to O.D.K.).

1. Dissect Lower Hemi-mandibles from Adult Mouse
<ol>
	<li>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.
	<ol>
		<li>OPTIONAL: Separate head from the rest of the body using a razor blade.</li>
	</ol>
	</li>
	<li>Remove skin from the lower lip to the neckline.</li>
	<li>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.</li>
	<li>Wedge the scalpel between the condyle of the jaw and the temporal mandibular joint and cut alongside the jaw muscle to detach the jaw.</li>
	<li>Remove all muscle, tendon, and ligament until only the bone remains.</li>
</ol>
2. Isolate the Incisor
<ol>
	<li>Using a #15 scalpel, carefully shave off the bone at a 45&#176;&#160;angle from the distal end to the proximal end of the membranous region. Use the tip of the scalpel to pick away any remaining bone including the bone fragments at the edge. Slowly start to pick away at the lingual cortical plate of the mandible, starting just below the 3rd molar.</li>
	<li>Continue until the incisor is clean, working carefully towards the area containing the CL.</li>
	<li>Remove inferior alveolar nerve bundle.</li>
	<li>Make a clean cut to first remove the bone and tissues proximal to the CL.</li>
	<li>Make a second cut distally with the scalpel, roughly below the 2nd molar. The proximal incisor region is then dissected out by inserting the scalpel between the bone and the medial surface of the proximal incisor. This must be carefully done, so as not to tear the tissue. The incision motion is proximal-distal. 
	ALTERNATIVE STEP: 
	Remove as much bone as possible along the area above the incisor. Once area is cleared, carefully remove entire incisor using a size 5 forceps to handle the enamel portion closest to CL region.</li>
	<li>Place dissected tissue (either isolated CL as in Figure 3E, or entire incisor as in alternative Step 2.5) in 2% collagenase in 1X PBS for 3-4 hr&#160;at 4 &#176;C in a low adherence plate. For 1-10 incisors, use 100 &#956;l per well in a 12-well plate. For greater than 10 incisors, use 500 &#956;l per well in a 6-well low adherence plate.</li>
</ol>
3. Microdissect the Labial Cervical Loop
<ol>
	<li>Remove CL region from the collagenase and place in cold DMEM/F12 media.</li>
	<li>Pull gently at the lower end of the CL using either size 5 forceps or an insulin syringe (1 cc, 28 G &#189;); the mesenchyme will fall apart while the epithelium remains intact. The CL and the adjacent epithelium that extends laterally as a &#8220;wing-like&#8221; structure will be visible.</li>
	<li>Remove the apical bud from the adjacent epithelium by making a V-shaped incision on either side, and immediately place in cold DMEM/F12 on ice.</li>
	<li>Repeat for all CLs, collect in 1.5 ml microfuge tube.</li>
	<li>Spin down microfuge tube with specimens, remove media, replace with 100 &#956;l cell detachment solution.</li>
	<li>Incubate tissues in cell detachment solution for 30 min at 37 &#176;C.</li>
	<li>Dissociate the tissues to produce a single cell suspension by gently pipetting up and down using a 1,000 &#956;l low-adhesion pipette tip. Cells can also be placed through a sterile cell strainer to achieve dissociation.</li>
	<li>Count cells with hemocytometer, plate on tissue culture plastic or other desired material.</li>
</ol>
4. Primary Culture of DESCs
<ol>
	<li>Plate cells in a 6-well plate at 1-6 x 104 cell/ml in DESC media, which consists of DMEM/F12 supplemented with mouse EGF recombinant protein at a concentration of 20 ng/ml, FGF recombinant protein at a concentration of 25 ng/ml, 1X B27 supplement, and 1% antibiotic solution (penicillin, 100 U/ml, streptomycin, 50 &#956;g/ml).</li>
	<li>Grow cells plated on a 6-well plate at 37&#160;&#176;C in 5% CO2 in 1 ml of DESC media. Do not disturb initial cultures for 5 days after plating to allow cell adhesion and colony formation.</li>
	<li>For the first media change, replace half of the volume (500 &#956;l) of old media with half volume (500 &#956;l) of new media. Check colonies under microscope when changing media.</li>
	<li>After the first media change, continue to change media (1-2 ml) every 2 days. Check colonies under microscope when changing media.</li>
</ol>
5. Passage DESCs
<ol>
	<li>Aspirate media and wash cells 3x&#160;with pre-warmed PBS.</li>
	<li>Add pre-warmed 0.25% Trypsin-EDTA in saline solution and incubate at 37 &#176;C for 10-15 min&#160;for the DESCs to detach.</li>
	<li>Add DESC media to neutralize the trypsin, gently pipette along tissue culture plate to collect the cells, and place in a 15 ml conical tube.</li>
	<li>Spin down cells at 800 x g for 2 min.</li>
</ol>

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</ol>

The authors have no conflicts of interest to disclose.

Epithelial cells were first successfully cultured over 40 years ago21-24, and more recently, successful isolation of epithelial stem cells25-27 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 ...

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 advance6-13.
All rodents possess continuously growing incisors whose growth is fueled by stem cells, making these teeth an accessible model system to study adult stem cell regulation. Labeling experiments in the 1970s10,11, followed by genetic lineage tracing experiments8,9,12,14, have demonstrated that DESCs reside in the proximal region of the incisor. The stem cell progeny in the epithelial compartment on the labial side move out of the putative niche, known as the labial cervical loop (CL), and contribute to a population of cells called transit-amplifying (T-A) cells (Figure 1). Specifically, DESCs reside in the outer enamel epithelium (OEE) and stellate reticulum (SR)8,9,14, and the inner enamel epithelium (IEE) gives rise to the T-A cells that progress through a limited number of cell cycles and then move distally along the length of the incisor (Figure 1). The differentiating ameloblasts in the mouse incisor continue to move distally along the incisor at a remarkable rate of approximately 350 &#181;m in a single day15,16. As they move, the cells differentiate into mature ameloblasts and stratum intermedium (SI) cells. After depositing the full thickness of enamel matrix, many of the ameloblasts undergo apoptosis, and the remaining cells shrink in size and regulate enamel maturation17. The lineage of the other cell types in the labial CL, such as the SR, is less clear, and data regarding the stem cells in the mesenchyme18 and in the lingual CL are only beginning to emerge.
Using the mouse incisor model, a number of groups have worked to elucidate the genetic pathways and cell biological processes involved in natural stem cell-based organ renewal. However, the labial CL contains a relatively small number of cells, estimated to be about 5,000 per mouse incisor, which makes working with primary cells challenging. Therefore, efforts have been made to culture and expand these cells in vitro6,16,19,20&#160;in order to open new doors to experimental approaches that are not achievable in vivo. The most recent study has demonstrated that these cells can both self-renew and differentiate into amelogenin expressing cells when cultured13. Here, we describe and demonstrate a method for the reliable and consistent culture of cells from the mouse labial CL.

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isolation and culture of dental epithelial stem cells from the adult mouse incisor

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

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;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.

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...

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.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

The overall goal of this procedure is to obtain cells for culture from the mouse incisor, labial cervical loop in a reliable and consistent manner. This is accomplished by first dissecting the lower Hemi mandible from the adult mouse and removing the bone covering the cervical loop region. The second step is to isolate the cervical loop region and separate the meen kind from the epithelium.Next, the cervical loop is micro dissected. The final step is to dissociate then culture, the isolated epithelial cells. Ultimately, this video is used to show the correct procedure for successful isolation of cells from the labial cervical loop.Demonstrating the procedure will be myself, postdocs Kirsten, SEL, and Jimmy Hu and ing Lee, a visiting faculty member here in the Klein laboratory. After euthanizing the animal, remove the skin from the lower lip to the neckline. Then use a scalpel to make an incision between the two lower incisors, which are loosely connected.Upon applying pressure, the mandible will be split into two halves. Next, WeCh the scalpel between the condyle of the jaw and the temporal mandibular joint, and cut alongside the jaw muscle to detach the jaw after word. Remove all the muscles, tendons, and ligaments until only the bone remains.Now carefully shave off the bone at a 45 degree angle from the proximal end to the distal end of the membranes region. Use the tip of the scalpel to remove any remaining bone, including the bone fragments at the edge. Then slowly start just below the third molar to remove the lingual cortical plate of the mandible.Continue to clean. Working carefully around the area containing the cervical loop. Make a clean cut to first remove the bone and tissues proximal to the cl.Then make a second cut distally with the scalpel roughly below the second molar. After that, remove the inferior alveolar nerve bundle. The proximal incisor region is then dissected out by inserting the scalpel between the bone and the medial surface of the incisor.This must be done carefully so as not to tear the tissue. Then place the dissected tissue in 2%collagenase in one XPBS for three to four hours at four degrees Celsius in a low adherence plate for one to 10 incisors. Use 100 microliters per well.In a 12 well plate, and for greater than 10 incisors, use 500 microliters per well in a six well low adherence plate. In this procedure, remove the CL region from the collagenase and place in cold D-M-E-M-F 12 media. Next, pull gently at the lower end of the CL using either forceps or an insulin syringe.The mesenchyme will fall apart while the epithelium remains intact. Then the CL and the adjacent epithelium that extend laterally as a wing leg structure will be visible. Remove the apical bud from the adjacent epithelium by making a V-shaped incision on either side and place it in cold DMMF 12 on ice.Immediately repeat for all CLS and collect them in a 1.5 milliliter micro fuge tube. Next, spin down the micro tube with the specimens. Remove the media and replace it with 100 microliters of cell detachment solution.Then incubate the tissues in cell detachment solution for 30 minutes at 37 degrees Celsius. After word dissociate the tissues to produce a single cell suspension by gently pipetting up and down. Using a 1000 microliter low adhesion pipette tip count cells with a hemo cytometer subsequently plate the cells on tissue culture, plastic, or other desired material.Now plate the cells in a six well plate at one to six times 10 of the fourth cells per milliliter. In DESC media Grow the cells plated at 37 degrees Celsius in 5%carbon dioxide. Do not disturb the initial cultures for five days after plating to allow for cell adhesion and colony formation for the first media change.Replace 500 microliters of the old media with 500 microliters of new media. Check the colonies under a microscope when changing the media after the first media change change with one to two milliliters of media every two days and check the colonies under a microscope When ready to passage cells, aspirate the media and wash the cells three times with prewarm PBS. Then add the PREWARM 0.25%tripsin EDTA in saline solution and incubate at 37 degrees Celsius for 10 to 15 minutes for the DSCs to detach.Next, add DESC media to neutralize the trypsin. Gently pipette along the wall of the tissue culture plate to collect the cells and place them in a 15 milliliter conical tube After word spin down the cells at 800 Gs for two minutes. Shown here is the visualization of the cervical loop.Using a fluorescent reporter mouse, the bone is relatively autofluorescent. Removal of the bone exposes the cervical loop and the rest of the epithelium, which can then be easily excised. All structures of the cervical loop can be easily visualized with the reporter gene.This figure shows the representative results of the successful colony formation in vitro. This image of DSCs is taken under the phase contrast microscope after plating and culture for seven days. Tight colony formation indicates successful epithelial isolation with no mesenchymal contamination.After 10 days of incubation, colonies are larger in size, and the cells have a typical epithelial cobblestone morphology. After watching this video, you should have a good understanding of how to isolate and microdisect the cervical loop and culture dental epithelial stem cells from the adult mouse incisor.

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Research

JoVE Journal

Biology

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