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. Author manuscript; available in PMC: 2011 Aug 15.
Published in final edited form as: Dev Biol. 2010 Jun 4;344(2):795–799. doi: 10.1016/j.ydbio.2010.05.511

Induction of human keratinocytes into enamel-secreting ameloblasts

Bingmei Wang a,*, Liwen Li a,*, Shengrong Du a,*, Chao Liu b, Xin Lin a,b, YiPing Chen a,b,, Yanding Zhang a,
PMCID: PMC3010760  NIHMSID: NIHMS212555  PMID: 20678978

Abstract

Mammalian tooth development relies heavily on the reciprocal and sequential interactions between cranial neural crest-derived mesenchymal cells and stomadial epithelium. During mouse tooth development, odontogenic potential, that is, the capability to direct an adjacent tissue to form a tooth, resides in dental epithelium initially, and shifts subsequently to dental mesenchyme. Recent studies have shown that mouse embryonic dental epithelium possessing odontogenic potential is able to induce the formation of a bioengineered tooth crown when confronted with postnatal mesenchymal stem cells of various sources. Despite many attempts, however, postnatal stem cells have not been used successfully as the epithelial component in the generation of a bioengineered tooth. We show here that epithelial sheets of cultured human keratinocytes, when recombined with mouse embryonic dental mesenchyme, are able to support tooth formation. Most significantly, human keratinocytes, recombined with mouse embryonic dental mesenchyme in the presence of exogenous FGF8, are induced to express the dental epithelial marker PITX2 and differentiate into enamel-secreting ameloblasts that develop a human-mouse chimeric whole tooth crown. We conclude that in the presence of appropriate odontogenic signals, human keratinocytes can be induced to become odontogenic competent; and that these are capable of participating in tooth crown morphogenesis and differentiating into ameloblasts. Our studies identify human keratinocytes as a potential cell source for in vitro generation of bioengineered teeth that may be used in replacement therapy.

Keywords: bioengineered tooth, human keratinocyte stem cell, mouse dental mesenchyme, FGF8

INTRODUCTION

Multiple signaling molecules, including BMPs, FGFs, WNTs, and SHH, have been implicated in tooth formation. These molecules mediate the sequential and reciprocal interactions between the dental epithelium and mesenchyme, regulating tooth morphogenesis and differentiation. Both dental epithelial and mesenchymal tissues are indispensable for tooth development (McCarrol and Dahlberg, 1934; Koch, 1967). Classic tissue recombination studies have shown that in mice, prior to embryonic day 12 (E12), dental epithelium initially acquires the odontogenic potential, that is, the capability to instruct tooth development in tissues of non-dental origin. Subsequently, odontogenic potential shifts to the dental mesenchyme, enabling the latter to direct non-dental epithelium of embryonic source to form a tooth (Yoshikava and Kollar, 1981; Mina and Kollar, 1987; Lumsden, 1988). Interestingly, this odontogenic inducing capability appears to reside only in the molar mesenchyme but not in the incisor mesenchyme (Song et al., 2006). Tooth development undergoes various stages, with the formation of tooth crown through the differentiation of dentin-secreting odontoblasts and enamel-producing ameloblasts. Ameloblasts are lost via their transformation and apoptosis upon tooth eruption (Abiko et al., 1996), making it impossible to repair or replace lost/damaged enamel in erupted teeth.

Recently, stem cell based tissue engineering has given hope to the possibility of regeneration or de novo formation of tooth structures (Ikeda and Tsuji, 2008). While bioengineered tooth germs from mouse embryonic molar cells are able to develop into fully functional tooth replacement in the lost tooth region of adult mice (Ikeda et al., 2009), using a patient’s adult stem cells to develop bioengineered replacement teeth would be preferable to the use of embryonic tissues, and is the ultimate goal of regenerative dental medicine. Various postnatal mesenchymal stem cells, including adult human dental pulp stem cells and bone marrow stromal cells, have been shown capable of differentiating into odontoblasts and creating mineralized dentin (Gronthos et al., 2000; Miura et al., 2003; Ohazama et al., 2004; Seo et al., 2004; Sonoyama et al., 2006). While it has been shown that cell lines derived from mouse embryonic dental epithelium are able to support and participate in the formation of bioengineered teeth when combined with mouse embryonic dental mesenchyme (Komine et al., 2007), attempts to direct postnatal stem cells to differentiate into enamel-secreting ameloblasts have not been successful thus far. Sine the demand for replacement teeth is significant and ameloblast stem cells do not exist, the identification and application of postnatal stem cells as the epithelial component for tooth regeneration represent major challenges for regenerative therapy.

Mammalian skin consists of dermal and epidermal components. The epidermis is formed of multiple layers of keratinocytes at different stages of differentiation, such that that basal layer, which directly contacts the basement membrane, contains proliferating cells and keratinocyte stem cells. Keratinocyte stem cells give rise to a population of transient amplifying cells that undergo a limited number of divisions, differentiate, and move up within the epidermis to maintain epidermis turnover. The accessibility of these cells, and the ease with which they can be cultured has prompted wide clinical use of human keratinocytes, particularly in the reconstitution of the epithelial component, following burns and chronic wounds, and in gene therapy (Alonso and Fuchs, 2003). In this study, we investigate whether confluent epithelial sheets of cultured human keratinocytes could be used as an epithelial component for the production of bioengineered teeth using tissue recombination and ex vivo culture approaches. We demonstrate that when recombined with mouse embryonic dental mesenchyme, human kertinocyte epithelial sheets are able to support the differentiation of dental mesenchymal cells into dentin-secreting odontoblasts. Furthermore, addition of FGF8 growth factor to theese tissue recombinants induces human keratinocytes to express the dental epithelial marker PITX2 and to differentiate into enamel-secreting ameloblasts in a human-mouse chimeric tooth crown.

MATERIALS AND METHODS

Isolation and culture of human keratinocytes

Primary human keratinocytes were isolated and cultured following the protocol described previously with some modifications (Jones and Watt, 1993). Briefly, circumcised human foreskins from 5 – 12 years old children were processed immediately after collection. Tissues were sliced into small pieces (0.1 cm × 2 cm), and epidermal layer was separated from dermis after overnight incubation with 2 U/ml Dispase at 4°C. Epidermis was further incubated with 0.25% trypsin/0.05%EDTA at 37°C for 15 min, dispersed into a single cell suspension after the cornified layer was discarded. About 2 × 106 suspended epidermal cells were plated onto a 6-cm cell culture dish in Keratinocyte Serum-free Medium (KSFM) (Gibco). Unattached cells were discarded by medium replacement after 6 hr in culture. Attached cells were continuously cultured in KSFM, formed colonies and grew to confluence. In such culture condition, cells could be grown for 5 to 6 passages. Passage 2 or 3 cells were used for recombination experiments. Epithelial cell sheets were removed from the dishes by slight shaking, sliced into about 1-mm2 size, and then stored in culture medium on ice for subsequent tissue recombination experiments.

Tissue recombination and kidney capsule culture

Tissue recombination and mouse kidney capsule culture were carried as described previously (Song et al., 2006). Briefly, mandibular molar tooth germs were isolated from E13.5 mouse embryos, incubated in 2.25% trypsin and 0.75% pancreatin in PBS on ice for 10 min, and dental epithelia removed with fine forceps. A piece of mouse dental mesenchyme was recombined with a piece of human keratinocyte cell sheets. Recombination of the mouse dental mesenchyme with the previously separated dental epithelium served as controls. Recombinants were cultured in Trowell type organ culture for 24 hr prior to being subjected to subrenal culture in immune-compromised adult male mice. For bead implantation, agarose beads (Bio-Rad) were soaked with FGF8 (125 μg/ml, R&D Systems), BMP4 (200 μg/ml, R&D Systems) or SHH (250 μg/ml, R&D Systems), respectively, as described previously (Zhang et al., 2000), and implanted into tissue recombinants in organ culture. Recombinants together with beads were subjected to subrenal culture after 24 hrs in organ culture. Samples were harvested 4-week after subrenal culture and processed for histological analysis and immunohistochemical staining.

Histology, immunochemical staining, in situ hybridization, and RT-PCR

For histological analysis, recombinant samples were fixed in 4% paraformaldehyde (PFA), decalcified in 10% ethylenediaminetetraacetic acid for 1-week, dehydrated in graded ethanol, and embedded in paraffin. Sections were made at 7-μm, and were processed for hematoxylin/eosin staining or Azan dichromic staining for histological analysis, and subjected to immunohistochemical staining using an antigen recovery technique. Cultured human primary keratinocytes at 30% confluence were subjected to immunocytochemical analysis. The following antibodies were used: anti-human MHC I and anti-mouse MHC I (Biolegend); anti-human ameloblastin, anti-human FGF8, and anti-human MMP-20 (Santa Cruz); anti-human CD29, anti-human K15, and anti-human K19 (Neomarkers). Immunostaining procedures followed the instructions by the manufacturers. For negative controls, the primary antibodies were omitted. Procedures for section in situ hybridization and information for the human PITX2 and FGF8 probes have been described previously (Lin et al., 2007). Standard RT-PCR analysis was performed to examine PITX2 expression in culture human keratinocytes and in human keratinocyte epithelial sheet in tissue recombinants using the primers (upper: 5′CGGCCGCAGTTCAATGGGCT-3′; and lower: 5′-TGAGACTGGAGCCCGGGACG-3′). GAPDH was included as control (upper: 5′-ATGGGGAAGGTGAAGGTCG-3′; and lower: 5′-GGGTCATTGATGGCAACAATA-3′).

Human embryos

Surgically terminated human embryos were provided by the Hospital for Women and Children of Fujian Province. Use of human embryonic tissue was approved by the Ethics Committee of Fujian Normal University. To obtain human tooth germs at a late differentiation stage, the premolar tooth germs were isolated from embryonic tissue, grafted under the kidney capsule of adult nude mice, and remained in subrenal culture for 8 weeks prior to harvest. Samples were fixed in 4% PFA, decalcified, dehydrated, embedded in paraffin, and sectioned at 7-μm for in situ hybridization and immunohistochemical analyses.

RESULTS AND DISCUSSION

Cultured human skin keratinocytes have potential in many prominent clinical applications, such as therapeutic reconstruction of the epithelial component, following burns, chronic wounds and ulcers (Alonso and Fuchs, 2003). We investigated whether human skin keratinocytes represent an applicable source for generation of bioengineered teeth and enamel production. We isolated human keratinocyte stem/progenitor cells from freshly collected foreskins that were surgically circumcised from 5–12 years old children, as described previously (Jones and Watt, 1993; also see “Materials and Methods’). Primary human keratinocytes that attached to the culture dishes exhibited extensive cell proliferation capability and formed colonies (Fig. 1), suggesting that these are keratinocyte stem/progenitor cells; this was further confirmed by identification of several molecular markers using specific antibodies, including K15, K19 and CD29 (Fig. 1) (Hotchin et al., 1995; Jones, 1993; Lyle et al., 1998; Michel et al., 1996).

Figure 1.

Figure 1

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Culture of primary human keratinocytes. (A) Colony formation of cultured primary human keratonicytes 5-day after plating. (B) Colony formation of cultured human keratinocytes 10-day after plating. (C) Cultured human keratinocytes stained positive for K15. (D) Cultured human keratinocytes stained positive for K19. (E) Cultured keratinocytes stained positive CD29. (F) Negative control for immunocytochemical staining. Scale bars: 100 μm (A, B); 20 μm (C, D, E, F).

To obtain keratinocyte epithelial sheets for tissue recombination experiments, isolated human keratinocyte stem/progenitor cells were grown to confluence. Confluent epithelial sheets were carefully removed from the dishes, sliced into about 1-mm2 squares, and then processed for tissue recombination. A piece of epithelial sheet was recombined with an E13.5 mouse molar mesenchyme, shown to possess odontogenic potential (Lumsden, 1988; Mina and Kollar, 1987; Song et al., 2006). Recombinants were cultured in Trowell type organ culture for 24 hr prior to being subjected to subrenal culture for 4-week. Histological analysis demonstrated the formation of a whole tooth crown-like structure consisting of human and mouse tissues in 25% cases (6/24) (Fig. 2A). Close examination revealed the presence of well-formed dentin; however keratinocyte-derived epithelial cells failed to differentiate into elongated ameloblasts (Fig. 2A). Immunohistochemical studies using specific antibodies against human or mouse MHC I antigens confirmed the human origin of the epithelial component (Fig. 2B) and the mouse origin of the dental pulp (Fig. 2C), respectively. Human keratinocyte-derived epithelium is thus able to support mouse embryonic dental mesenchyme to form tooth crown-like structures. However, epithelial cells do not have ameloblastic differentiation capacity under this condition.

Figure 2.

Figure 2

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Formation of human-mouse chimeric tooth crown. (A) A tooth crown-like structure formed in a recombinant of human keratinocytes and mouse E13.5 molar mesenchyme. Arrows point to the flat epithelial cells. (B) Anti-human MHC I antibody stained the epithelial cells (arrows) of human origin in a human-mouse chimeric tooth crown. (C) Anti-mouse MHC I antibody stained the dental pulp cells of mouse origin. D, dentin; O, odontoblasts; P, pulp. Scale bar: 100 μm.

Next, we surveyed several key growth factors for their potential in inducing human keratinocytes to differentiate into ameloblasts. Among them are SHH and BMP4 that are expressed in the differentiating ameloblasts of mouse and human teeth (Dassule et al., 2000; Lin et al., 2007), and FGF8 which is expressed in both odontoblasts and ameloblasts of developing human teeth throughout early and late developmental stages (Lin et al., 2007; supplemental Fig. 1). Beads soaked with each of these growth factors were implanted into tissue recombinants of human keratinocyte cell sheet and mouse molar mesenchyme, respectively. Tissue recombinants were subsequently subjected to subrenal culture for 4-week. Neither SHH nor BMP4 induced ameloblastic differentiation, while BMP4 induced significant amount of bone formation instead of tooth in the recombinants (Supplemental Fig. 2). In contrast, while the success rate of tooth formation in the recombinants remained the same in the presence of FGF8 (125 μg/ml) (41/146), FGF8 in fact induced the keratinocyte-derived epithelial cells to become elongated and to deposit enamel in 13 cases of the 41 formed teeth (Fig. 3A). Immunohistochemical assays demonstrate the presence of ameloblastin and MMP-20, specific markers for differentiated ameloblasts, in the elongated epithelial cells and the enamel (Fig. 3B, 3C), confirming ameloblastic differentiation. Furthermore, the positive staining for FGF8 in the differentiated ameloblasts mimics the endogenous FGF8 expression pattern in developing human teeth (Fig. 3D).

Figure 3.

Figure 3

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Differentiation of human keratinocytes into enamel-secreting ameloblsts in the presence of FGF8. (A) A recombinant tooth crown showed elongated ameloblasts (arrow in insert) and deposition of enamel and dentin. (B) Specific staining of human ameloblastin in the ameloblasts (arrows). (C) Immunostaining of human MMP-20 in the ameloblasts (arrow) and enamel (asterisks). (D) Immunostaining of FGF8 in the ameloblasts. Note the odontoblasts and dental pulp cells of mouse origin stained negatively. A, ameloblast; D, dentin; E, enamel; O, odontoblasts; P, pulp. Scale bar: 100 μm.

Pitx2 represents one of the earliest molecular markers in the dental epithelium in mice, and is involved in the specification of odontogenic epithelium (Mucchielli et al., 1997; St. Amand et al., 2000). Pitx2 is expressed in the presumptive dental epithelium before tooth formation and throughout the entire tooth developmental process, and its initial expression is induced by FGF8 (St. Amand et al., 2000). Mutations in Pitx2 result in tooth agenesis in mice and humans (Semina et al., 1996; Lin et al., 1999; Lu et al., 1999). We have previously demonstrated that human developing tooth germ exhibits a PITX2 expression pattern identical to that observed in mice (Lin et al., 2007). Thus we wondered if FGF8 specifies human keratinocytes to odontogenic fate by activating PITX2 expression. We therefore examined PITX2 expression in human keratinocytes in tissue recombinants. RT-PCR assays revealed an absence of PITX2 expression in cultured human keratinocytes, but the activation of PITX2 in tissue recombinants in the presence of FGF8 at day 4 and day 6 of culture (Fig. 4). In situ hybridization further confirmed the expression of PITX2 in the epithelial sheet of human keratinocytes in tissue recombinants (Fig. 4B). These results further confirm an odontogenic fate commitment and differentiation of the human keratinocytes induced by the mouse embryonic dental mesenchyme and FGF8.

Figure 4.

Figure 4

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Activation of PITX2 in the human keratinocytes in the recombinants in the presence of FGF8. (A) RT-PCR assays show the expression of PITX2 in 11-week old human embryonic premolar germ (11W), absence of PITX2 in human keratinocytes before tissue recombination (KSC) and in the tissue recombinant in the presence of FGF8 2-day (2d) after recombination, the expression of PITX2 in the tissue recombinants in the presence of FGF8 4-day (4d) and 6-day (6d) after tissue recombination. (B) In situ hybridization shows the expression of PITX2 in the human keratinocyte aggregate in a recombinant with the mouse dental mesenchyme in the presence of FGF8. (C) Negative control of in situ hybridization for PITX2 using sense probes.

It is interesting to note that in the developing human embryo, deciduous teeth begin to develop during the 6th week of gestation, but it is not until the 28th week that dental epithelium starts to differentiate into enamel-producing ameloblasts (Schoenwolf et al., 2009). However, in the human-mouse chimeric tooth crown produced in this study, differentiated ameloblasts and deposited enamel are found within 4-week under the subrenal culture. This is likely due to the more rapid differentiation of mouse odontoblasts, which in turn induces earlier ameloblastic differentiation of the human keratinocytes.

In vitro generation or assembly of an implantable bioengineered human tooth from stem cells will have extremely significant impact on dental practice. A bioengineered tooth will require two populations of stem cells: one for the mesenchymal component and one for the epithelial component. In this regard, identification and characterization of suitable sources of dental stem cells are essential. Several types of mesenchymal stem cells have been isolated from adult human teeth, including dental pulp stem cells (Gronthos et al., 2000), stem cells from periodontal ligament (Seo et al., 2004), and stem cells from apical papilla (Sonoyama et al., 2006). While their capacity to induce or to participate in de novo tooth development is not evident, these stem cells are indeed capable of differentiating into odontoblasts and specified periodontal cells. However, isolation of dental epithelial stem cells remains impossible, because enamel epithelial cells do not exist in human teeth, since ameloblasts surrounding the tooth crown undergo apoptosis during tooth eruption. Thus alternative sources of postnatal stem cells are needed as the epithelial component for in vitro construction of a bioengineered tooth. Despite substantial effort to identify such a source, there is no evidence so far that any type of postnatal stem cell is odontogenic competent as an epithelial component, and can participate in tooth morphogenesis, subsequently giving rise to differentiated enamel-secreting ameloblasts. Human keratinocytes have wide clinic applications due to their nature as easily accessible cells and their ability to be culture and rapidly expanded. In this study, we demonstrate that human keratinocytes are not only odontogenic competent and capable of participating in tooth crown morphogenesis, but that they can also differentiate into ameloblasts and produce enamel. The odontogenic process apparently can be initiated in postnatal stem cells of non-dental origin when proper odontogenic signals are provided. Among these putative odontogenic signals, FGF8 appears to be critical. In mice, Fgf8 is expressed in the dental epithelium during early tooth development and is responsible for the induction of Pitx2 that is involved in the specification of dental epithelium (Mucchielli et al., 1997; St. Amand et al., 2000). However, Fgf8 is never expressed in the dental mesenchyme and its expression ceases in the developing tooth around the bud stage in mice. In contrast, FGF8 is continuously expressed in both the dental epithelium and dental mesenchyme in the human developing tooth germ (supplemental Fig. 1). The latter observation explains the additional requirement for FGF8 for the induction of human keratinocytes into ameloblastic differentiation by mouse dental mesenchyme. FGF8 very likely functions together with odontogenic signals emanating from the mouse dental mesenchyme to specify the odontogenic fate of human keratinocytes through activation of PITX2. Similar to the endogenous expression pattern of FGF8 in developing human tooth germs, we observed FGF8 expression in the differentiated ameloblasts of human origin in the human-mouse chimeric tooth. However, Fgf8 expression is not activated in the odontoblasts of the mouse origin (Fig. 3). While identification of the odontogenic signals emanating from mouse dental mesenchyme possessing odontogenic potential warrants future investigation, FGF8 apparently represents an essential signaling molecule required for odontogenic fate determination and differentiation of the human keratinocytes. While we are far from understanding the identity, properties, and functional mechanisms of the odontogenic signals, and just as far from in vitro construction of an applicable bioengineered tooth using solely postnatal stem cells, our studies nevertheless identify human keratonictes an appropriate cell source for generating bioengineered whole tooth crown for future dental replacement and regenerative therapy.

Supplementary Material

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Acknowledgments

The authors thank Dr. Fiona Inglis of Tulane University for comments and proofreading of the manuscript. This work was supported by grants to Y. Z. from the Ministry of Science and Technology of China (“973” Project: 2005CB522700, 2010CB944800), National Natural Science Foundation of China (30771132), and the Ministry of Education of China (20093503110001), and the NIH grants (DE15123 and DE12329) to Y.C.

Footnotes

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