The significance of “niche” or “microenvironment” has been often emphasized, especially in developmental biology, but it was used as an abstract argument and an obstacle against analytical biology. However, recent technical advancement and the movement of “tissue engineering” have made the concept of niche a much more practical theme. Several recent studies indicate that a microenvironment cancels the already-made program in foreign cells and allows them to take its own program [19,20,21].

Our previous study reported that five clonal emtg lines expressed sets of genes specific for different developmental stages of the dental epithelium and cells of emtg-2 expressed genes specific for preameloblasts [18]. The present study demonstrated that reconstructed germs developed into teeth at 100% when emtg-2 cells in the epithelial compartment were mixed with EC (E16.5) (> 1:0.5). The epithelial compartment in the improved germs revealed the pattern of gene expression which is observed at the initiation stage corresponding to E11.5–12.5 [1,2]. Enzymatic separation and recombination of the epithelial and mesenchymal compartments set the developmental clock back to the initiation stage [22,23,24,25,26]. These observations suggest that the developmental program in emtg-2 cells was canceled by the microenvironment created in the germs. Then, the microenvironment might coordinate two populations of epithelial cells and mesenchymal cells and allow them to restart odontogenesis from the initiation stage.

The in vitro model on culture insert is useful, although it has several weaknesses that need to be improved upon. One of them is the extra-formation of enamel knots or Shh-expression spots. After three days in culture, reconstructed germs with cells of emtg-2 and EC expressed the pattern of gene expression similar to that of bud stage, and three Shh-expression spots were detected in the germs. Another weakness is poor formation of basement membrane. The amelogenin expression was appropriate in the germ implanted under kidney capsule [18]. However, the boundary between epithelial and mesenchymal compartments became less prominent when the germs were cultured on inserts instead of implanting under kidney capsule. It is possible that the formation of the basement membrane is not fully progressing and secreted amelogenin protein spreads into the area of mesenchyme.

In our previous study, germs reconstructed with only emtg-2 cells and MT developed into tooth at near 60% when they were implanted under kidney capsule [18]. In the present study, however, germs reconstructed with only emtg-2 cells and MT developed into no teeth when they were cultured on inserts. In general, the environment under kidney capsule is known to be superior to those of any tissue culture dishes. It is reasonable to think that the undefined factors under kidney capsule help the reconstructed germs to cancel the program in emtg-2 cells and allow them to restart odontogenesis from the initiation stage.

Germs reconstructed with mixed epithelial cells (emtg cells: EC = 1:0.5) and MT developed into teeth at varied efficiency: 42% (emtg-1), 100% (emtg-2), 67% (emtg-3), 0% (emtg-4) and 58% (emtg-5). A similar but not identical tooth construction was observed when germs were reconstructed with cells of each emtg line and MT and implanted under kidney capsule [18]. In both studies, MT were regularly prepared from embryos at E16.5. In addition, germs prepared with EC at different ages developed into to teeth at varied rates; 50% (E14.5), 86% (E15.5) and 100% (E16.5) (Table 3). These observations indicate that the age of tissue (cells) and cell lines in reconstructed germs has critical influence on tooth construction. Heterochronological recombination of dissociated epithelial and mesenchymal cells disturb tooth organogenesis [22], although the organogenesis progressed when tissue architectures of both epithelial and mesenchymal compartments were preserved [39].

In order to investigate how reprograming of emtg-2 cells is induced, germs were prepared with two pellets of epithelial cells. The results indicate that EC had direct effects on emtg-2 cells. If this is the case, more EC in germs would give rise to higher tooth construction. As expected, the rate of successful tooth reconstruction was decreased as the mixing ratio was reduced from 1:1 to 1:0 (emtg-2: dental epithelial cells). Germs reconstructed with emtg cells and MT might have some difficulty to create the microenvironment which coordinates epithelial and mesenchymal components to restart odontogenesis from the first stage of a series of reciprocal epitheo-mesenchymal interactions [1,2]. “EC give emtg cells direct signals, diffusible factors and/or physical contact, which are highly expressed at E16.5, then the epithelial compartment becomes competent to communicate with the mesenchymal compartment. To identify the signals would further help to understand odontogenesis.”

CD-1 mice (Charles River Japan, Yokohama, Japan) were maintained in the experimental animal facility of Tokyo University of Science. They were kept under a 12:12 h light: dark cycle at 22–24 °C. Standard laboratory feed (MR standard, Nousan LTD, Yokohama, Japan) and tap water were given ad libitum. Mice care and handling conformed to the NIH guidelines for animal research. The Institution Animal Care and Use Committee approved the experimental protocols.

The medium was a 1:1 mixture of Dullbecco’s modified Eagle’s medium and Ham’s nutrient mixture F12 (DMEM/F12, Sigma, St. Louis, MO, USA) supplemented with heat-inactivated fetal calf serum at 10% (10% FCS v/v, Sigma, St. Louis, MO, USA), penicillin (31 ìg/mL, Sigma, St. Louis, MO, USA), streptomycin (50 ìg/mL, Sigma, St. Louis, MO, USA), insulin (10 ìg/mL, Sigma, St. Louis, MO, USA) and transferrin (10 ìg/mL, Sigma, St. Louis, MO, USA). Cell lines were maintained in the medium and passaged at 10% on 100 mm dishes (FALCON; Becton Dickinson Labware, Franklin Lakes, NJ, USA).

For histology, samples were fixed in 4% formaldehyde at 4 °C overnight and dehydrated with sucrose for frozen sections, and embedded in O.C.T. Compound (Sakura Finetek, Torrance, CA, USA), or dehydrated with ethanol for paraffin embedding. Samples were cut into 10 ìm sections. Sections were stained with hematoxyline and eosin, or incubated with antibodies. The primary antibodies used were anti-GFP (1:500, Santa Cruz Biotechnology, Inc., Heidelberg, Germany) and anti-amelogenin (1: 100, Hokudo Co., Ltd., Sapporo, Japan) rabbit polyclonal antibodies. After several washes with PBS containing 0.1% Tween-20 (Sigma, St. Louis, MO, USA), sections were incubated with secondary antibodies; Alexa Fluor 488 goat anti-rabbit IgG (1:500, Invitrogen Corp., CA, USA) and Alexa Fluor 594 goat anti-rabbit IgG (1:500, Invitrogen Corp., CA, USA) for 1.5 h at room temperature. Negative controls were incubated without primary antibodies. Nuclei were stained with DAPI.

The method was developed and described elsewhere [22]. In brief, dental germs of lower first molars were dissected from embryos at day (E) 14.5, 15.5 and 16.5 and incubated in PBS containing 1000 U/mL dispase II (SANKOJUNYAKU CO. LTD., Tokyo, Japan) for 15 min at room temperature, and the epithelia were separated from the mesenchymal tissues with a fine needle. Epithelial fractions were treated with 100 U/mL collagenase type I (Worthington Biochemical Co., Lakewood, NJ, ISA) for 20 min at 37 °C. After several washings with PBS, they were treated with 0.5 × trypsin-0.53 mM EDTA (Sigma, St. Louis, MO, USA) for 5 min at 37 °C and dispersed. emtg cells and dental epithelial cells were mixed at ratios of 1:1, 1:0.75, 1:0.5, 1:0.25 or 1:0. Suspensions containing epithelial cells were centrifuged to make pellets. The total epithelial cell number was 3 × 10⁴ cells/germ. Tooth germs were reconstructed with the pellets of mixed epithelial cells and with dental mesenchymal tissues prepared from embryos in 30 ìL Cell matrix type I-A (Nitta Gelatin Inc., Osaka, Japan) and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. On the following day, the reconstructed tooth germs were implanted under kidney capsule or cultured on cell culture inserts (FALCON; Becton Dickinson Labware, Franklin Lakes, NJ, USA) for seven days (Figure 14).

Plat-E packaging cells were transiently transfected with pMXs-Ig modified at 5'LTR to LTR/CMV (gifted from Dr. Murakami, Tokyo University of Science), using LipofectamineTM2000 Reagent (Invitrogen). After 48 h, the transfection solution was added to each emtg cell line cultured on 24-well plates.

Samples were embedded in O.C.T. Compound (Sakura Finetek) without fixation and frozen. Cryosections (10 ìm) were made and mounted on FRONTER slides (MATSUNAMI, Osaka, Japan). Digoxigenin-labeled probes for specific transcripts were produced with the digoxigenin RNA labeling kit (Roche, Mannheim, Germany) and prepared by PCR with primers designed using published sequences (Table 7 and [22]). Resultant bands were subcloned into pGEM T-easy vector (Promega, Madison, WI) and sequenced to confirm the sequence identity. Hybridization was carried out at 57 °C for 16 h with hybridization buffer containing 50% formamide, 0.01 M Tris-HCl (pH 7.4), 1 mM EDTA, 0.1% SDS, 0.6 M NaCl, 1× Denhardt’s solution, 10% dextran sulfate, and 400 ìg/mL yeast tRNA. The final wash was in 0.2× SSC at 57 °C for 1 h. Samples were incubated for 2 h with alkaline phosphatase-conjugated sheep anti-digoxigenin Fab fragments (Roche) at 1:1000, washed and processed for colorimetric detection using NBT/BCIP Stock Solution (Roche).]