The Differentiation Potentiality Of The Mouse Embryonic Stem Cells To Dental Epithelial Cells

Teeth are highly mineralized organs resulting from sequential and reciprocal interactions between the oral epithelium and the underlying cranial neural crest-derived mesenchyme. Tissue-engineered teeth need dental epithelial and mesenchymal cells. In recent years, various tissue-derived adult stem cells have already demonstrated the potential to give rise to dental mesenchymal like cells, such as dental pulp stem cells (DPSCs), adult bone marrow cells, skin dermal multipotent stem cells, hair follicle dermal papilla, et al. However, to date, there are few reports on the successful transformation of cells into dental epithelial cells. Although bone-marrow-derived cells can be reprogrammed to form ameloblast-like cells, simultaneous differentiation of 1 cell type into the cells of 2 different embryonic lineages has restricted the utility of this approach. Oral mucosal epithelium can be a potential source of dental epithelial cells; however, using 1 post-natal autologous palatal epithelium limits the use of this approach. These shortcomings necessitated the search for other cell sources of ameloblasts using simple and convenient methods. Embryonic stem cells (ESCs) have attracted great interest recently because they can serve as a potentially unlimited supply of cells that can be differentiated toward specific lineages of any cell type in the body. ESCs can give rise to epithelial cells of the skin, lung, thymic, et al. However, there have been few studies and little knowledge about whether mESCs can differentiate into dental epithelial cells. The present study provides a better understanding of the mechanism of ameloblasts, and insights into the selection of candidate cells for dental epithelial cells. The contents of the present study are as follows:1. Cell culture and Characterization of mESCs.Objective: To culture ESCs R1 cell line and CGR8 cell line and confirm the identity of mESCs. Methods: MESCs were cultured in the absence of feeder cells for 4 days. The culture medium was Dulbecco's modified Eagle medium supplemented with 15% heat-inactivated fetal bovine serum (FBS; Gibco), 1% penicillin/streptomycin, 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), and 1000 U/mL leukemia inhibitory factor (LIF; Chemicon, USA). Alkaline-phosphatase (AP) staining was performed according to the manufacturer's recommendations (Millipore) and EB formation was achieved. For teratoma formation, approximately 2×106 ESCs were injected into the subcutaneous pockets of 6-week-old nude mice. At 4 weeks after ESC injection, xenografted masses were observed and dissected. Results: ES cells have the typical morphological characteristics: the nest-like colony with clear edge and smooth surface; the boundaries between cells is not clear and the single cell volume is small. Next we evaluated the AP activity of mESCs; enzyme assay results revealed high AP activity. In vitro methods involving EB formation are simple and widely used because this method created suitable conditions for the differentiation into the cells of all 3 germ layers. Teratomas were formed 4 weeks after injection in vivo. HE staining and immunohistochemistry analysis of the germ-layer differentiation markers revealed that the teratomas displayed the derivatives of all 3 embryonic germlayers. Conclusion: ESCs possess an intrinsic self-renewal ability and can differentiate into numerous types of functional tissue cells.2. Differentiation of ameloblasts serum-free conditioned medium (ASF-CM) induced mouse embryonic stem cells.Objective: To explore the possibility of mESCs for odontogenic differentiation induced by ASF-CM. Methods: Two in vitro culture techniques have been developed to stimulate the differentiation of ESCs, which include the formation of embryoid bodies (EBs) and direct culture of ESCs as monolayers. Odontogenic differentiation environment was mimicked by indirect co-culture method. The indirect co-culture using conditioned medium was performed as we previously described. In brief, mESCs with or without EB step at a density of 1×104 cells/ mL were plated in 6-well culture plates. Experiments were carried out in ASF-CM for 14 days. The conditioned medium was renewed every 24 h. Results: After treated with ASF-CM, cobblestone-like cells, which resembled dental epithelial cells, were observed at the periphery of the plated mEBs and in the mESC monolayer. Moreover, the proportion of these cobblestone-like cells increased with incubation time. As early as 7 days of treatment with ASF-CM, mEBs expressed ameloblast-specific proteins such as CK14, AMBN, and AMGN. However, DSPP and DMP-1 were not expressed. Furthermore, the level of expression at 14 days was higher than that at 7 days. Protein expression of ameloblasts markers were determined by immunofluorescent staining and Western blot. After culturing the cells for 14 days in the differentiation-inducing media, the expression of ameloblast-specific proteins such as cytokeratin (CK)14, ameloblastin (AMBN), and amelogenin (AMGN) was markedly higher in mESCs obtained with embryoid body (EB) formation than in mESCs obtained without EB formation. Western blot analysis further confirmed the the existence of CK14, AMBN and AMGN in two experimental groups after 14-day co-culture, suggesting that treated mESCs with EB stage can enhance odontogenesis in vitro. We observed that immunocompromised mice implanted with induced murine EBs (mEBs) showed tissue regenerative capacity and produced odontogenic epithelial-like structures, whereas those implanted with mSCE monolayer cells mainly formed connective tissues. Furthermore, the epithelial-like tissues formed by ASF-CM-treated mEBs positively expressed CK14, AMBN and AMGN. Normal developing mandible samples were as positive control. When labeled with CFSE and transplanted with 1dpn mice tooth germ cells, the induced mESCs with EBs could produce mineral deposits after four weeks incubation (66.7% of the transplanted cell pellets). However, in control group, undifferentiated mES with tooth germ cells in vivo still formed the teratomas. Conclusion: Thus, for the first time, we report that ASF-CM provides a suitable microenvironment for inducing mESC differentiation along the odontogenic epithelial cell lineage. This result has important implications for tooth tissue engineering.3. Differentiation of tooth germ cell conditioned medium (TGC-CM) induced mouse embryonic stem cells.Objective: To explore the possibility of mESCs for odontogenic differentiation induced by TGC-CM. Methods: It remains to be elucidated whether TGC-CM can induce mESC differentiation towards odontogenic lineage. Changes in cell morphology, RT-PCR analysis, immunofluorescence staining and western blotting analyses were performed. In vivo differentiation potential of TGC-CM-induced mESCs were also performed. Results: When cultured in TGC-CM, cobblestone-like cells were not observed in the 2 groups. Furthermore, the cultured cells appeared damaged and cell proliferation activity was low when TGC-CM was used. RT-PCR analysis indicated that TGC-CM induced mEBs expressed only DMP-1 and weakly expressed CK14 after treatment for 7 and 14 days, respectively. The results of mESC culture without EB formation in TGC-CM were similar with those of EB-mediated culture. In vivo those implanted with induced mEBs or mSCE monolayer cells mainly formed connective tissues and teratomas were formed in few implants. Conclusion: Collectively, these results indicate that ASF-CM is a better inducing medium than TGC-CM for promoting mESC differentiation towards ameloblast lineage.