The mouse incisor contains valuable label-retaining cells in its stem cell niche. We have a novel way to unbiasedly detect and quantify the label-retaining cells; our study used EdU labeling and a 3D reconstruction approach after PEGASOS tissue clearing of the mandible.
The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the proximal tissues of incisors give rise to progeny that will differentiate into ameloblasts, odontoblasts, and pulp fibroblasts. These cells are crucial in supporting the sustained turnover of incisor tissues, making the murine incisor an excellent model for studying the homeostasis of adult stem cells. Stem cells are believed to contain long-living quiescent cells that can be labeled by nucleotide analogs such as 5-ethynyl-2´-deoxyuridine (EdU). The cells retain this label over time and are accordingly named label-retaining cells (LRCs). Approaches for visualizing LRCs in vivo provide a robust tool for monitoring stem cell homeostasis. In this study, we described a method for visualizing and analyzing LRCs. Our innovative approach features LRCs in mouse incisors after tissue clearing and whole-mount EdU staining followed by confocal microscopy and a 3-dimensional (3D) reconstruction with the imaging software. This method enables 3D imaging acquisition and non-biased quantitation compared to traditional LRCs analysis on sectioned slides.
The continuously growing mouse incisor is an excellent model for studying adult stem cells1. The epithelial (labial and lingual cervical loop) and mesenchymal stem cells (between the labial and lingual cervical loop) that reside on the proximal side of the incisor differentiate into ameloblasts, odontoblasts, and dental pulp cells. This unique process provides a source of cells for compensation of tissue loss and turnover2. Though several stem cell markers such as Sox2, Gli1, Thy1/CD90, Bmi1, etc. have been identified in vivo for the subsets of adult stem cells in mouse incisors, they are inadequate in representing the stem cell populations when used alone1,3,4,5. Visualizing long-living quiescent cells by nucleotide analog DNA labeling and retention could provide unbiased detection for most subsets of adult stem cells6. Further, this approach is useful among many stem cell identification methods7 for understanding cell behavior and the homeostasis of dental stem cell populations3,8. While the dividing stem cells would lose their DNA labeling after a considerable chase, the putative quiescent stem cells retain their DNA label, deeming them label-retaining cells (LRCs)6. DNA labeling and retention by non-dividing stem cells will mark and locate the putative adult stem cells in their niches.
Over the past years, thymidine analog 5-bromo-2′-deoxyuridine (BrdU) labeling replaced the cumbersome, time-consuming, and high-resolution microscopy incompatible 3H-thymidine DNA labeling method for cell proliferation assays6,9. In recent years, the 5-ethynyl-2´-deoxyuridine (EdU) labeling technique has been increasingly used over BrdU. This pattern emerged due to several reasons. First, the BrdU method is slow and labor-intensive. User conditions are variable, and it is unable to preserve the ultrastructure in specimens (due to DNA denaturation). Likewise, the BrdU method loses the antigenicity of cells, thus making it inefficient for downstream functional analyses and assays such as the co-localization experiments and in vivo stem cell transplantation3,7,9,10,11,12. BrdU is also a teratogen, which is not suitable for labeling LRCs in embryonic development6. Also, the BrdU method is inefficient when used in the whole-mount specimens. The disadvantages are low penetration of antibodies in the deep part of specimens or the requirement of a long antibody incubation period for deep penetration13. EdU labeling escapes the steps of denaturing specimens, thus preserving the ultrastructure. This feature is advantageous for downstream functional analyses such as co-localization experiments and stem cell transplantation11,12. Also, EdU labeling is highly sensitive and rapid; specimen penetration is high due to the use of rapidly absorbed and smaller-sized fluorescent azides for detecting EdU labels through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry)14.
Another increasingly applied DNA labeling method is the use of engineered transgenic mice. These mice express histone 2B green fluorescent protein (H2B-GFP) controlled by a tetracycline-responsive regulator element5,14. After feeding mice with tetracycline chow/water for a 4-week to 4-month chasing period, the GFP fluorescence will diminish in cycling cells and only LRCs retain the fluorescence6. The advantage of this method is that the labeled LRCs can be isolated and remain viable for cell culture or downstream functional analyses6,7. Some studies reported inaccurate labeling of quiescent stem cells when chased for long-term use. This result was due to a leaky background expression from the H2B-GFP strain and not the appropriate tetracycline-regulated response15.
Moreover, most literature in the past used the LRCs detection mainly on sectioned slides, which are two dimensional and often erroneously biased in showing the accurate location and number of LRCs. The approach displayed incorrect angles for sections of complex tissue structures16. The other method was to obtain 3D images from serial sections and perform post-image reconstructions. These steps were inaccurate because of image distortion from variations in each serial section due to compressed or stretched sections, resulting in missing information16,17,18. The method was also laborious and time-consuming.
To facilitate the whole-mount imaging of LRCs, samples need to be made clear while the fluorescence be well maintained. Current tissue clearing techniques can be classified into three major categories: organic solvent-based tissue clearing techniques, aqueous reagent-based tissue clearing techniques, and hydrogel-based tissue clearing techniques17,19. The polyethylene glycol (PEG)-associated solvent system (PEGASOS) has been recently developed. This approach renders nearly all types of tissues transparent and preserves endogenous fluorescence, including hard tissues such as bone and teeth20. The PEGASOS method has advantages over other tissue clearing methods, especially in clearing tooth and bone materials. Most other methods could only partially clear hard tissues, have long processing times, or require costly reagents21. Also, the PEGASOS method can efficiently preserve endogenous fluorescence over other methods.
This literature led us to create a new method for cell study. We combined the LRCs detection advantages of EdU labeling with the most superior 3D whole-mount imaging of tissue-cleared specimens; samples were processed with advanced polyethylene glycol (PEG)-associated solvent system (PEGASOS) tissue clearing technique15. Hard tissue transparency enabled us to reconstruct the 3D signal of LRCs fluorescence in vivo without breaking the teeth or mandible, creating a more accurate way to visualize and quantify LRCs.
In this study, we provide an innovative guide to visualize LRCs in the mouse incisor. We made a 3D visual approach to determine the location and quantity of LRCs within the mouse incisor stem cell niche. This project used EdU labeling, PEGASOS tissue clearing techniques, and confocal microscopy. Our method of EdU labeling the LRCs on whole-mount tissue and the use of a cleared and transparent specimen overcomes both the limitations of traditional sectioned slides and other disadvantageous DNA labeling methods. Thus, our technique will be suitable for studies on stem cell homeostasis requiring LRCs detection, especially on hard tissues. The protocol can be equally advantageous to those focusing on stem cell homeostasis in other tissues and organs.
All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) for Texas A&M University College of Dentistry.
1. Preparation of the EdU labeling cocktail
2. Preparation of the mice and EdU solution
3. Sample preparation by trans-cardiac perfusion (postnatal 53-day-old mice after the 6-week chase period)
NOTE: The mouse liver should turn pale after successful trans-cardiac perfusion.
4. Tissue clearing of the mandible using the PEGASOS technique
NOTE: A 15 mL or 50 mL conical tube as per the volume of tissues can be used to keep the samples ready for treatment in each step. Samples are processed at 37 °C shakers (~100 rpm) from steps 4.2 to 4.7. Use polypropylene-based plastic containers that are resistant to organic solvents to avoid melting the plastic. Alternatively, glassware can be used.
CAUTION: The PEGASOS tissue clearing technique uses toxic solutions such as Quadrol, polyethylene glycol (PEG), benzyl benzoate (BB), MMA500, etc. Appropriate PPE is required to avoid potential exposures.
5. Confocal imaging of the tissue-cleared mandible
6. Image processing, 3D image reconstruction, and quantification of label-retaining cells by creating a surface, segmentation of the Region of Interest (ROI), masking the ROI, and creating spots
NOTE: We used Imaris (Bitplane 9.0.1) for image processing and 3D reconstruction, but similar image processing steps can be conducted using other software suites (e.g., ImageJ/Fiji, 3D slicer, Avizo, Arivis, Amira, etc.).
After EdU labeling and the PEGASOS tissue clearing process (Figure 2), the transparent mandible was obtained as shown in our image (Figure 3B). We compared the modified sample to a normal mandible without a tissue clearing process (Figure 3A). The transparent mandible (Figure 3B) with EdU labeling was subjected to confocal imaging. We focused on the incisor apex showing the stem cell niche as shown in (Supplemental Figure 1). The optical section of the incisor showed LRCs in the stem cell niche of the incisor apex in the XY plane (Figure 4A). We reconstructed a 3D image of an incisor apex showing EdU+ label-retaining quiescent stem cells in both the epithelial (green) and mesenchymal (red) stem cell niche (Figure 4B). EdU+ cells were transferred into spots for quantification that comparably overlapped with the mesenchymal LRCs (Figure 4C). Figure 4D shows the spots created only for mesenchymal LRCs. Figure 4E shows the spots created only for epithelial LRCs. We then completed the quantification of epithelial and mesenchymal LRCs (Figure 4F).
Figure 1: EdU injection protocol to label LRCs. Wild-type (WT) mice were injected with EdU starting at P5. The injections lasted for 7 consecutive days until P11. Next, the mice underwent a chase period of 6 weeks. We harvested them on postnatal day 53. Please click here to view a larger version of this figure.
Figure 2: Workflow of the protocol. Mice were injected with EdU for 7 consecutive days, and then placed in a chase period for 6 weeks. The mandibles were harvested and fixed after trans-cardiac perfusion. Next, the mandibles were decalcified and subjected to the tissue clearing steps of decalcification, decolorization, whole-mount EdU staining, de-lipidation, dehydration, and refractive index (RI) matching. The imaging for the cleared mandibles was performed under a confocal microscope followed by data analysis. Please click here to view a larger version of this figure.
Figure 3: Images of a postnatal 53-day-old WT mouse mandible before tissue clearing (A) and after tissue clearing (B). Please click here to view a larger version of this figure.
Figure 4: An optical section of the incisor showing LRCs in the stem cell niche of the incisor apex in the XY plane (A). A 3D image reconstruction of epithelial and mesenchymal LRCs in mouse mandibular incisors toward the apex (B). Overlapping mesenchymal LRCs (red) with created 3D spots (gray) (C). Spots created only for mesenchymal LRCs (D). Spots created only for epithelial LRCs (E). Quantification results for epithelial and mesenchymal LRCs (F). Bar: 300 µm in A and 150 µm in B-E. Please click here to view a larger version of this figure.
Preparation of EdU Labeling Cocktail |
Preparation of stock solutions (aqueous) |
TBS (10x) 1 M, pH 7.6 |
CuSO4 (100x), 0.4 M |
Sulfa-Cyanine 3 Azide (100x), 300 µM in DMSO |
Sodium Ascorbate (10x), 0.2 g/mL in H2O |
PBST (0.1% Triton X-100 in PBS, v/v) |
Preparation of EdU Labeling Cocktail |
Add the following reagents in order: |
Tris-buffered saline (100 mM final, pH 7.6) |
CuSO4 (4 mM final) |
Sulfa-Cyanine 3 Azide (3 µM final) |
Sodium Ascorbate (100 mM final, freshly made for each use) |
Table 1: Preparation of the EdU labeling cocktail
Supplemental Figure: Please click here to download this File.
Multiple doses of injections (BrdU, EdU) are usually used on growing neonatal mice to label proliferating cells as much as possible1,6,13. The chasing period is considered a critical step regarding the renewal rate of tissues6,13. The mouse incisor renews itself around every month. This trait allows researchers to set the chasing period to 4 weeks or longer4,5,22. Our 6-week chase period could label the stem cells in mesenchymal and epithelial (labial and lingual cervical loop) compartments. This area included some of the progenitor populations in TACs (Transit-Amplifying Cells) from both epithelial and mesenchymal compartments. A longer chase period would be desirable in showing true label-retaining cells that reside exclusively in the epithelial and mesenchymal stem cell niches.
There are multiple critical steps in the tissue processing and clearing stages for this protocol. The first note is for the tissue perfusion and fixation step. Mice incisors contain pulp tissues that have a rich supply of blood vessels and contain a high amount of blood tissues. Thus, the heparin PBS perfusion should be started as soon as possible after the mice are deeply anesthetized and restrained. The reason is to avoid blood clot formation that leads to autofluorescence19. Care should be taken to avoid over-fixation with PFA, which may lead to insufficient transparency and yellowish discoloration of the tissue after clearing. Excessive discoloration lowers the signal quality in the samples during imaging19. The active trans-cardiac perfusion step is preferred over passive immersion fixation of organs/tissues, which will rapidly fix the tissues to avoid loss of endogenous fluorescence in the tissue clearing steps. In passive fixation, the areas deeper in the tissue may remain less transparent; the samples may not have satisfactory imaging outcomes19. Proper removal of muscles from the mandibles allows for proper visualization of structures that interest researchers. Further, effective removal prevents autofluorescence disturbances from impacting muscle tissues. Proper decalcification is indispensable in hard tissue clearing. Care should be taken to adequately decalcify tissues by adjusting the EDTA immersion time as per the size and mineral content of the samples. EDTA concentration is critical to avoid over shrinkage of the tissues. Therefore, the concentration should be chosen as per the samples and experiments in question16,19. Likewise, a decolorization step is critical to adequately remove the remaining heme to reduce the autofluorescence. Inadequate de-lipidation time can result in less transparent tissue and is not advised. Generally, de-lipidation for hard tissues such as mice mandibles can be done from 4-6 h in 30% and 50% tB solution and for 1 day in 70% tB solution20. The incubation time depends on the size of the sample and its lipid content. The time can be prolonged to ensure complete delipidation19,20. Individual laboratories can adjust the timing as per their requirements without lessening the minimum time required for de-lipidation.
The most common problem in tissue clearing techniques involves the inadequate transparency of tissue caused by improper tissue processing and clearing steps. This situation leads to difficulty in obtaining clear images, hindering proper visualization of the fluorescent-labeled cellular or subcellular structures. Troubleshooting the inadequately transparent tissues should be done by ensuring each critical step is done correctly. This practice should start from tissue perfusion and fixation steps to the tissue clearing steps. The properties of each tissue or organ are different. Hence, the tissue processing and clearing steps should be optimized to obtain satisfactory clearing results19.
The imaging of cleared tissue samples can be completed with a confocal laser scanning microscope (CLSM) or a light sheet fluorescence microscope (LSFM) depending upon the requirements and experimental question and availability of the equipment17. We used CLSM for imaging the mandible, which gives higher resolution imaging at higher magnification but takes a longer time compared to LSFM17,20. When high resolution and higher magnification are not a requirement, the fast light-sheet fluorescence microscope (LSFM) may be desirable19. LSFM is expensive and may not be easily available for regular labs. For confocal microscopy, choosing the right objective lenses with the right numerical aperture is critical for good imaging19,20. For large tissue samples, lower magnification objectives such as 10x with a small numerical aperture and a greater working distance may be appropriate19,21. For smaller tissue samples, higher magnification objectives such as 20x with a high numerical aperture and a smaller working distance may be desirable. Also, using an immersion oil with a matching refractive index to the clearing medium and an objectives lens is critical in avoiding image distortion and gaining clarity17,19. For imaging, several image processing and analysis software suites are available. While some of the software suites are free, others are expensive and may be unaffordable for individual labs. These imaging platforms generate massive files (in gigabytes) that require higher-end computer workstations for data visualization and quantification17,20,21.
Though quicker and easier to perform than other DNA labeling methods such as BrdU, there are limitations to detecting LRCs through EdU labeling in vivo compared to labeling LRCs with H2B-GFP transgenic mice. First, it is difficult to distinguish the LRCs of the epithelial origin or mesenchymal origin. H2B-GFP labeling can be used in a tissue-specific, promoter-driven, tetracycline activator that can specifically label the quiescent stem cells in various tissues. In the case of mice incisors, the H2B-GFP method can specifically label the stem cells from the epithelium or mesenchyme4,22. However, the tissue clearing and 3D image construction methods described in this protocol also apply to H2B-GFP fluorescence-labeled LRCs, providing flexibility and a variety of options. Our work enables the labeling and characterizing of LRCs according to scientific needs. The H2B-GFP method requires transgenic mice production and cross-breeding with other strains, which is time-consuming and costlier than EdU labeling. The other limitation of EdU labeling with the tissue clearing method is that the specimens cannot be used further for downstream functional analyses.
This protocol is advantageous to investigators that do not require tissue-specific labeling and want quicker results from labeling quiescent stem cells. This method can be modified to use for cell lineage tracing; when incorporated with Cre-driven fluorescence labeling of stem/progenitor cells, our method obtains more details on progeny location and more accurate quantitation/contributions23.
The authors have nothing to disclose.
We thank Meghann K. Holt for editing the manuscript. This study was supported by NIH/NIDCR grants DE026461 and DE028345 and the startup funding from the Texas A&M School of Dentistry to Dr. Xiaofang Wang.
0.5 M EDTA | Sigma Aldrcih | E9884 | |
20 × Objective/NA 0.9 | Leica | 507702 | |
50 mL Falcon Centrifuge Tubes | Falcon | 352070 | |
BD PrecisionGlide Needle | BD | REF 305111 | |
Bezyl benzoate (BB) | Sigma Aldrcih | 409529 | |
Bitplane 9.0.1 | Imaris | ||
BRAND cavity slides | Millipore Sigma | BR475505 | |
C57BL/6J mice | Jackson Laboratory | Strain #:000664 | |
Circulation Pump | VWR | 23609-170 | |
CuSO4 | Sigma Aldrcih | 451657 | |
DMSO | Sigma Aldrcih | D8418 | |
EdU | Carboynth | NE08701 | |
Heparin | Miiilipore Sigma | H3149 | |
Imaging System | Olympus | DP27 | |
LAS X Software | Leica | ||
Olympus Stereo Microscope | Olympus | SZX16 | |
Paraformaldehye | Sigma Aldrich | P6148 | |
PBS | Sigma Aldrich | P4417 | |
PEGMMA500 | Sigma Aldrich | 447943 | |
Quadrol | Sigma Aldrich | 122262 | |
Sodium Ascorbate | Sigma Aldrich | 11140 | |
Sulfa-Cyanine 3 Azide | Lumiprobe | D1330 | |
TBS-10X | Cell Signaling Technology | 12498 | |
TCS SP8 Confocal Microscope | Leica | ||
tert-butanol (tB) | Sigma Aldrich | 360538 | |
Triton X-100 | Sigma Aldrich | X100 |