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Efficient induction of functional ameloblasts from human keratinocyte stem cells
© The Author(s). 2018
Received: 3 February 2018
Accepted: 1 March 2018
Published: 2 May 2018
Although adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. In addition, whether the regenerated enamel retains a running pattern of prism identical to and acquires mechanical properties comparable with human enamel indeed warrants further study.
Cultured human keratinocyte stem cells (hKSCs) were treated with fibroblast growth factor 8 (FGF8) and Sonic hedgehog (SHH) for 18 h or 36 h prior to being recombined with E13.5 mouse dental mesenchyme with implantation of FGF8 and SHH-soaked agarose beads into reconstructed chimeric tooth germs. Recombinant tooth germs were subjected to kidney capsule culture in nude mice. Harvested samples at various time points were processed for histological, immunohistochemical, TUNEL, and western blot analysis. Scanning electronic microscopy and a nanoindentation test were further employed to analyze the prism running pattern and mechanical properties of the regenerated enamel.
Treatment of hKSCs with both FGF8 and SHH prior to tissue recombination greatly enhanced the rate of tooth-like structure formation to about 70%. FGF8 and SHH dramatically enhanced stemness of cultured hKSCs. Scanning electron microscopic analysis revealed the running pattern of intact prisms of regenerated enamel is similar to that of human enamel. The nanoindentation test indicated that, although much softer than human child and adult mouse enamel, mechanical properties of the regenerated enamel improved as the culture time was extended.
Application of FGF8 and SHH proteins in cultured hKSCs improves stemness but does not facilitate odontogenic fate of hKSCs, resulting in an enhanced efficiency of ameloblastic differentiation of hKSCs and tooth formation in human–mouse chimeric tooth germs.
Various efforts to develop techniques for human tooth regenerative therapy and replacement have been attempted for decades [1, 2]. Currently, bioengineering of a whole tooth crown from embryonic tooth germ cells appears to be the most successful approach for tooth regeneration in several animal models including mouse, rat, pig, and dog [3–10]. Impressively, it was reported that implantation of a bioengineered tooth germ, reconstructed from mouse embryonic dental epithelial and mesenchymal cells, into a lost tooth socket in the alveolar bone of adult mice could develop into a fully functional tooth [11–14], indicating the feasibility of future regenerative therapy in humans via implantation of bioengineered tooth germs. However, in practice, it is impossible to use embryonic cells for such clinical therapy. Thus, identification of adult cell sources, such as stem cells from adult tissue or induced pluripotent stem cells (iPSCs), for ex-vivo generation of implantable tooth germ is a prerequisite for the realization of human biotooth replacement therapy in the future.
Stem cell-based tissue engineering has been proven a prospective approach to repair or replace an injured tissue or organ. Adult bone marrow stem cells (bone marrow stromal cells) are the first adult cell source capable of participating in tooth formation when confronted with the mouse embryonic dental epithelium that possesses odontogenic inducing capability . At least five types of mesenchymal stem cells from adult human teeth have been isolated . Among them, dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), and stem cells from the apical papilla (SCAP) could generate dentin/pulp-like complexes in ex-vivo culture [17–19]. Although these adult dental stem cells do not possess either odontogenic inducing capability or competence to support tooth formation when confronted with embryonic dental epithelia , they remain promising stem cell sources for regeneration of tooth mesenchymal components. On the other hand, the postnatal dental epithelium-derived stem cells are more difficult to obtain due to ameloblastic apoptosis during tooth eruption. It was reported that subcultured epithelial cell rests of Malassez can differentiate into ameloblast-like cells and generate enamel-like tissues in combination with dental pulp cells at the crown formation stage . We and others have reported previously that nondental epithelia-derived human stem cells including human keratinocyte stem cells (hKSCs) [20, 22], gingival epithelial cells , and iPSCs , when recombined with either human or mouse embryonic dental mesenchyme, could support tooth formation and differentiate into enamel-secreting ameloblasts. However, less than 30% and 10% of these recombinant explants in subrenal culture formed teeth and produced enamel, respectively . Such low efficiency of ameloblastic differentiation prevents use of these human stem cells as realistic cell sources for tooth replacement therapy. In addition, whether hKSC-derived dental epithelia exhibit an unusual life cycle and whether the regenerated enamel acquires the unique physicochemical characteristics remain elusive and warrant further exploration.
Studies indicated that either FGF8 or SHH alone is sufficient to promote limb regeneration in amphibian . FGF8 or SHH is able to stimulate neurite outgrowth and cavernous nerve regeneration in vitro, respectively [26, 27]. In the tooth, FGF8 promotes cell proliferation and inhibits apoptosis in diastemal tooth epithelium, and revitalizes the tooth developmental program . In this study, we developed an approach that greatly enhanced the ratio of ameloblastic differentiation of hKSCs and formation of tooth-like structures in tissue recombinants. We further examined the developmental process of differentiation of the hKSC-derived dental epithelium and present evidence for rapid differentiation of human ameloblasts and production of regenerated enamel with intact prisms the same as normal enamel. Meanwhile, we observed an increasing tendency for mineralization effect with improved mechanical properties in the regenerated enamel as cultivation extends. Our results provide a significant advance toward future use of human adult stem cells to generate implantable tooth organ ex vivo by tissue-engineering approaches.
Culture of hKSCs and application of recombinant proteins
Circumcised human foreskins from children 5–12 years old were collected immediately after surgery from Fuzhou Children's Hospital in Fujian Province. Primary human keratinocytes were isolated and cultivated in Keratinocyte Serum-free Medium (KSFM; Gibco) according to the protocol described previously . Keratinocyte stem cells were characterized by cell surface markers as described previously . Recombinant human FGF8a (100 ng/ml; R&D Systems) and/or SHH (100 ng/ml; R&D Systems) proteins were applied to passage 3 hKSCs cultured in KSFM at 90% confluence in 10-cm culture dishes. These cells were continuously cultured for 18 h or 36 h prior to being used for subsequent tissue recombinant experiments or immunocytochemical assay.
Tissue recombination and subrenal capsule culture
Tissue recombination and mouse subrenal culture were carried out as described previously . Briefly, mandibular molar tooth germs dissected from E13.5 mouse embryos were incubated in 2.25% trypsin and 0.75% pancreatin in PBS on ice for 10 min and then dental epithelia were removed with fine forceps. Pieces of confluent hKSC sheets were recombined with E13.5 mouse dental mesenchyme to reconstruct human–mouse chimeric tooth germs . Agarose beads (Bio-Rad) soaked with FGF8 (125 ng/μl; R&D Systems) and/or SHH (250 ng/μl; R&D Systems), respectively, were implanted into tissue recombinants as described previously . BSA beads were used as negative control. Recombinant tooth germs were cultured in Trowell type organ culture for 24 h prior to being subjected to subrenal culture in immune-compromised adult male mice. Samples were harvested at various time points after subrenal culture and processed for histological analysis and immunohistochemical staining.
Histology, immunochemical staining, TUNEL assay, and western blot analysis
Molar tooth germs dissected from surgically terminated human fetuses of 12th-week, 16th-week, and 19th-week gestation were provided by Fujian Province Maternal and Child Health Hospital. Use of human embryonic tissues in this study was approved by the Ethics Committee of Fujian Normal University, Fuzhou, China, and use of animals was approved by the Animal Use Committee of Fujian Normal University. Human fetus tooth germs and harvested recombinant samples treated with FGF8 and SHH protein prior to tissue recombination were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C on a rotator. Calcified tissues were further decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 1 week prior to being processed for dehydration and paraffin embedding. Sections were made at 10 μm, and were subjected to hematoxylin/eosin staining or Azan dichromic staining for histological analysis, and to immunohistochemical staining by antigen recovery technique. The following antibodies were used: anti-human ameloblastin, anti-human K18, anti-human p63, anti-human K10, anti-human integrin-β1 (Santa Cruz Biotech, Inc.), anti-human amelogenin, anti-human Sp3 (Abcam), anti-human Sp6, and anti-human Msx2 (HPA). For negative controls, the primary antibodies were omitted. Immunostaining, immunofluorescence, and TUNEL assay (Roche) procedures followed the instructions of the manufacturers. For western blot analysis, cultured hKSCs were extracted with urea lysis buffer. Equal amounts of samples were electrophoresed on 12% SDS polyacrylamide gels and transferred to NC membrane (Millipore). Immunoreactions were performed with the specific primary antibodies as mentioned earlier, visualized with fluorescent secondary antibodies (LI-COR), and scanned on an Odyssey Clx Imager (LI-COR). Blot images were quantified by densitometric analysis with ImageJ software.
Scanning electronic microscopy
The surface morphologies of human tooth (adult and child), mouse molar, and human–mouse chimeric tooth crown specimens that were treated with both FGF8 and SHH were investigated using a scanning electron microscope (S-3400 N; Hitachi, Japan) with an acceleration voltage of 15 kV. Each specimen was cold mounted in resin, abraded with #1200 SiC paper, polished with 0.05 μm alumina powder, etched with 25% EDTA for 60 s, washed in distilled water, and ultrasonically degreased in acetone. The conductive Pt thin film around 5 nm thick was sputtered on each specimen before scanning electronic microscopy (SEM) analysis.
where E r and ν are the reduced elastic modulus and Poisson’s ratio, respectively, for the specimen under test, and E i (1140GPa) and ν i (0.07) are the corresponding parameters of the diamond indenter. The Poisson ratio, ν, was 0.3 for each tooth sample . The fused quartz standard sample was used to calibrate the area function of the nanoindenter .
Enhanced ameloblastic differentiation efficiency of cultured hKSCs in the presence of FGF8 and SHH
Success ratio of tooth formation and ameloblastic differentiation in tissue recombinants
Protein in culture (h)
Protein beads in recombinant
Number of recombinants
Number of tooth formations
Ratio of tooth formation (%)
Number of ameloblastic differentiations
Ratio of ameloblastic differentiation (%)
We next treated the cultured hKSCs with either FGF8 (100 ng/ml) or SHH (100 ng/ml) proteins, or both of them, for 18 h or 36 h before proceeding to tissue recombination and subrenal culture (Table 1). It is noteworthy that hKSCs treated with either FGF8 or SHH alone, or both of them, respectively, retain much more healthy morphology with a smaller and typical cobblestone-like cell shape by comparison with control cells treated with PBS exhibiting a bigger and flattened phenotype (data not shown). Histological examination revealed that in samples cultured for 18 h, although the ratio of tooth formation remained around 50% (5 out of 11 recombinant samples) in the presence of FGF8 protein alone or in the presence of both FGF8 and SHH (9 out 17 recombinants), all teeth formed in both conditions exhibited a 100% ratio of ameloblastic differentiation (Table 1). We then extended the protein-treated culture duration to 36 h, and the tooth forming rate increased to 69% (9/13) with enamel deposition in all cases of the tooth forming samples (Table 1). However, treatment of cultured hKSCs with FGF8/SHH for periods longer than 36 h (48 and 60 h) resulted in decreased tooth formation and reduced ameloblastic differentiation in tissue recombinants (data not shown). Our results, therefore, definitely demonstrated a dramatically enhanced efficiency of ameloblastic differentiation and tooth formation by application of FGF8 and SHH protein in construction of human–mouse chimeric tooth recombinants.
FGF8 and SHH improves stemness but not ameloblastic fate of cultured hKSCs
Since application of FGF8 and SHH proteins in cultured hKSCs could dramatically increase the efficiency of ameloblastic differentiation and tooth formation in the human–mouse chimeric tooth germ, we investigated whether application of these proteins would commit cultured hKSCs to the odontogenic fate through induction of MSX2, SP3, and SP6 expression. We examined effects of FGF8 and SHH proteins on the expression of these three transcription factors in cultured hKSCs using immunofluorescence. Our results revealed that in control cells MSX2 and SP3 proteins were detectable whereas SP6 was not detectable (Fig. 2a–i). However, expression profiles of SP6, as well as MSX2 and SP3, remained unchanged in cultured hKSCs that were treated either with FGF8 (Fig. 2b, f, j) or SHH (Fig. 2c, g, k) alone or as a combination (Fig. 2d, h, l) for 18 h or 36 h. These results suggested that enhanced efficiency of ameloblastic differentiation and tooth formation by application of FGF8 and SHH in cultured hKSCs prior to tissue recombination might not be associated with commitment of hKSCs to odontogenic fate by activation of transcription factors of odontogenic importance.
The developmental process of differentiation of hKSC-derived dental epithelium into functional ameloblasts
In our previous report, we identified the human origin of hKSC-derived dental epithelial component and the mouse origin of the dental pulp with specific antibodies against human or mouse MHC antigen, respectively, in chimeric teeth to show no contamination of the mouse dental epithelial tissue in the recombinant experiment . In the present study, we further recombined hKSCs with mouse dental mesenchyme genetically labeled with eGFP. Immunofluorescence studies indicated that no GFP-positive cells could be found in hKSC-derived ameloblasts that were marked with SP6 in chimeric teeth (Fig. 4J). In addition, we grafted E13.5 dental mesenchyme with removal of dental epithelium after enzyme treatment into nude mice for subrenal culture for 4 weeks as a further control. All 30 grafted samples either degenerated or formed tiny pieces of bone-like tissues (data not shown). These data provide more evidence to rule out the possibility of mouse dental epithelium contamination in the recombinant experiment.
Microstructure and mechanical characteristics of regenerated human enamel
Reciprocal heterotypic recombination of tissues of ectopic origin has been long used as a routine approach to study regulative interactions between tissue components in classical experimental embryology. Mammalian tooth development is dependent upon inductive interactions between epithelium and adjacent mesenchyme . Both epithelial and mesenchymal components in tooth germ are indispensable for tooth development [46, 47]. Sequential and reciprocal interactions between the stomadial epithelium and the cranial neural crest-derived mesenchymal cells regulate tooth morphogenesis and differentiation. Odontogenic potential represents an instructive induction capability of a tissue to induce gene expression in an adjacent tissue and to initiate tooth formation, whereas odontogenic competence indicates the capability of a tissue to respond to odontogenic inducing signals and to support tooth formation. Tissue recombination experiments between isolated mouse molar epithelial and mesenchymal tissues have demonstrated that, during early tooth development, odontogenic potential resides first in the dental epithelium and then shifts to the mesenchyme [48, 49]. At the prebud stages of development (before and at E11.5), the presumptive dental epithelium possesses the potential to induce tooth formation in nondental mesenchyme. In contrast, at the early bud stage (E12.5) the odontogenic potential has switched to the mesenchyme, and this odontogenic mesenchyme is able to instruct nondental epithelium to form tooth-specific structures [48–50]. Our previous report demonstrated that such potential is also conserved in human embryonic dental mesenchymal tissues that are able to induce nondental epithelial tissues, such as human keratinocyte, and able to participate in tooth formation . In-vitro bioengineering of primordial tooth germs represents a promising approach for tooth replacement therapy in the future . Either in-vitro or ex-vivo generation of an implantable biotooth germ should follow the principles of tooth development. Based on this concept, previous studies including ours have demonstrated that human epithelium-derived stem cells, including iPSC-derived epithelium-like tissue, could be induced to participate in tooth formation when confronted with mouse dental mesenchyme with odontogenic potential. However, the efficiency of the induced ameloblastic differentiation of these cells was relatively low, and can be an obstacle for using adult stem cells as an epithelial cell source to develop tooth replacement therapy. In this study, in comparison with our previous report in which around 30% of tooth formation and 10% of ameloblastic differentiation were obtained , we achieved 70% and 100% of tooth formation and ameloblastic differentiation, respectively, by application of two key growth factors, FGF8 and SHH, in cell culture and tissue recombination, demonstrating that in the presence of appropriate odontogenic signals an efficient induction of enamel-secreting ameloblasts from hKSCs could be achieved.
FGF8 and SHH have been demonstrated to play pivotal roles in mammalian tooth development. SHH acts as an autonomous signal to regulate dental epithelial cells to proliferate, grow, and differentiate into functional ameloblasts [31, 52]. FGF8 is responsible for the determination of tooth forming sites, induction of several tooth developmental genes, and initiation of tooth development . We did find that, in the present study, application of these two growth factors activated SP6 expression in addition to that of MSX2 and SP3 expression in the hKSC-derived dental epithelium in tissue recombinants. The similar phenotypes in mice lacking the Msx2, Sp3, or Sp6 gene, respectively, and their overlapping expression pattern during tooth development raise the possibility that these transcription factors reside or interact closely within a signaling cascade to regulate amelogenesis [35, 36]. Our study indicates that simultaneous activation of these three transcription factors could likely be necessary for initiation of ameloblastic differentiation in hKSCs of the chimeric tooth germ. However, our results also indicate that application of FGF8 and SHH in the cultured hKSCs did not alter the expression of MSX2, SP3, and SP6 but improved stemness of hKSCs. These data strongly suggest that the enhanced efficiency of ameloblastic differentiation in hKSCs is associated with an improvement of cultured hKSC stemness but not their ameloblastic fate.
In the developing human deciduous teeth, ameloblasts begin to differentiate around the 15th week and start to secrete and deposit enamel on the surface of dentin around the 18th week of gestation . It is not until 6 months after birth that ameloblasts undergo apoptosis when the tooth erupts. Of interest, in our study, the differentiation and enamel deposit of hKSC-derived ameloblasts in the chimeric teeth were completed within 4 weeks under subrenal culture and this rapidly generated enamel exhibited a microstructural pattern grossly identical to normally developed enamel. Despite the mechanical property evaluation of enamel structures by a nanoindentation technique revealing much lower values of hardness and elastic modulus for the regenerated enamel than those of adult human and mouse teeth, an increasing tendency for the mineralization effect with cultivation time was discovered in this study. This should be of a significant impact for future clinical practice, since implantable tooth primordia could grow rapidly in the patient oral cavity in a relatively short period of time and further mineralize and mature to attain effective hardness and elastic modulus to withstand mechanical force applied during food chewing.
We developed a process for efficient induction of enamel-secreting ameloblasts and rapid generation of enamel from hKSCs by treatment of hKSCs with both FGF8 and SHH proteins prior to recombination with mouse embryonic dental mesenchyme. FGF8 and SHH dramatically enhanced stemness of cultured hKSCs. Electron microscopic analysis and a nanoindentation test revealed the formation of intact prisms and an increasing tendency for the mineralization effect with cultivation time in the regenerated enamel. Our results provide an appealing idea for efficient induction of adult stem cells into enamel-secreting ameloblasts.
The authors thank Fuzhou Children's Hospital in Fujian Province for providing circumcised human foreskins.
This study was supported by grants from the National Natural Science Foundation of China (81771034, 81271102, 81570036).
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
XfH, C-PL, and YdZ were responsible for conception and experiment design. XfH, J-WL, XZ, JhZ, XL, and XxH were responsible for the preliminary data search, selection, and extraction. YnS, BmW, H-HC were responsible for data analysis. XfH, C-PL, and YdZ were responsible for assembly of data, data analysis, and manuscript writing. YpC was responsible for data interpretation and manuscript revision. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All mouse surgical procedures were approved by the Animal Care Committee at Fujian Normal University. KSCs were harvested from circumcised human foreskins from children 5–12 years old whose parents gave informed consent for the study.
Consent for publication
The authors declare that they have no competing interests.
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