Teeth form by reciprocal interactions between epithelial and mesenchymal tissues derived from the ectodermal cell layer of the developing embryo. Tooth formation initiates when the epithelial lining of the oral cavity thickens to form primary epithelial bands (named placodes), which correspond in position to the future teeth in the upper and lower jaws. The epithelium then invaginates into the underlying mesenchyme, which responds to signals from this epithelium, to form the dental papilla. This process initiates a cascade of molecular signals. This cascade of signals between the dental epithelium and mesenchyme results in differentiation of the cells and tissues that make up a tooth organ, and the ultimate formation of a mature tooth. Observations of teeth formed in teratomas, show us that teeth can form from germ cells. Teratomas are tumors derived from pluripotent germ cells, and when they form in the ovaries, these tumors often contain well-differentiated cells that make tissues and organs, including hair and teeth. This evidence shows us that with the right trigger, a cascade of cell differentiation and reciprocal signaling in germ cells, can result in the complete formation of a tooth organ. Studies of tooth formation from human embryonic stem cells will also help us to understand fate decisions of tooth-specific lineage in early odontogenesis, as well and epithelial/mesenchymal interactions required in the development of tooth and other organs. We hypothesize that given suitable conditions, human embryonic stem cells (hESC) can commit to dental specific lineage cells. The odontogenic potential of these cells could progress once a reciprocal communication with mesenchyme is established, ultimately deposit dentin and enamel matrix proteins. Our specific aims are as follows: 1) to direct hESCs differentiation to dental epithelial lineage cells. In this first stage of tooth formation, we will determine the factors required for the formation of dental epithelial lineage cells, which can then direct dental mesenchymal differentiation as the cascade of events that result in tooth formation begins. 2) To determine the factors in the dental mesenchyme responsible for inducing the differentiation of dental epithelial lineage cells into ameloblasts. This second stage of differentiation is important for continued tooth formation. These experiments will help us to understand the signals required for maturation of the dental epithelium into ameloblasts, which are the cells responsible for tooth enamel formation. Our ultimate goal in these experiments is determine how teeth can differentiate from stem cells, and to use this knowledge to replace missing teeth in humans. In this seed grant we will take the first step toward this goal, to discover what directs the differentiation of dental lineage cells. The tooth organ model will help us to better understand epithelial-mesenchymal interactions in organ development.
Statement of Benefit to California:
There is currently much interest in bioengineering teeth, because dental decay and tooth loss constitutes an important health issue. In today’s market, the cost of each tooth replacement for Californians is around $2500. Currently, replacement of teeth requires prosthetic strategies including dental bridges, dentures and metal post implants covered by crowns. These prosthetic replacements must be replaced periodically, and there is no doubt that natural teeth are in all ways superior to these man-made materials. An ultimate goal in the use of stem cell research would be to promote the formation of new teeth to replace missing teeth, in the same way that permanent teeth replace the primary teeth of formed in early childhood. Of additional importance in this study is to understand cell fate decisions that direct the formation of organ-specific epithelial and mesenchymal tissues toward tooth formation. In other words, what factors direct germ cells to differentiate into the epithelial and mesenchymal cells of a tooth organ? The tooth is a particularly interesting model of organ development, forming a simple organ system from ectodermally derived epithelial and neurocrest cells. These cells continue to interact and influence each other’s differentiation as they progress down separate developmental pathways. If teeth can form following an initial ”triggering” event that starts the cascade of cell differentiation and interaction that results in tooth formation, then similar mechanisms may be sought in development of other organ systems. In summary, funding of this seed grant will allow us to pursue this unique research on tooth formation, helping us to understand the mechanisms of tooth formation, as well enhancing our basic knowledge of cell fate decisions in organ development. These studies could help Californian’s enjoy a life with functional teeth at all ages.
SYNOPSIS: The aim of this proposal is to examine the differentiation of hES cells into dental epithelial cells and to determine how dental mesenchyme promotes further differentiation of dental epithelium to ameloblasts. The ultimate goal would be to develop novel methods for replacement with new teeth. To monitor hES differentiation to dental epithelial cells, ectodermal cells derived from ES cells will be marked with an Fgfr2-EGF construct. The odontogenic potential of these cells in response to different conditions, will be determined by ondotogenic initiation marker expression and will be further tested on mouse E11.5-14 mesenchyme. A second aim will focus on determining the factors in dental mesenchyme responsible for inducing differentiation of dental epithelial lineage cells into enamel producing cells (ameloblasts). hES cells will be differentiated to neural crest cells using conditions established for primate ES cells. Cells will be sorted by anti-Ncx-1 antibody. FGF8 and BMP4 will be used to differentiate these cells into dental mesenchyme. Epithelial and mesenchymal cells can then be combined to investigate steps in ameloblast development. INNOVATION AND SIGNIFICANCE: This proposal addresses what is likely to be an understudied area. It is novel in it's focus on generation of methods for forming new teeth. About 20% the US population has congenitally missing teeth and a large number of the aging population experience tooth loss. Bridges and implants are commonly used to replace these lost teeth, but must be replaced periodically. Bioengineering of implantable teeth from stem cells represents an ideal approach for tooth replacement in the near future. Although attempts have been made to produce dental tissues/organs from stem cells including mouse ES cells, little is known about the specific potential and molecular basis of stem cells in stem cell-based tissue engineering of tooth structures. This proposal aims to direct differentiation of human ES cells into dental epithelial cells as well as dental mesenchymal cells, which is innovative. The second part of the proposal will also determine the factors in the dental mesenchyme responsible for inducing the differentiation of dental epithelial cells into ameloblasts. Successful completion, even partly, would make contribution to the field of dental biology. Knowledge gained from these studies would provide important insight in tooth regeneration in humans and may be an area where hES cells may provide new avenues for therapy. STRENGTHS: The PI, Dr. Pamela Den Bensten, is a dentist researcher with extensive research experience in enamel biology, which is one of the strengths. Strengths also include the insight of the PI, the excellent research environment at UCSF, the original and imaginative nature of the science involved, and the established methodologies in the PI’s lab, such as isolation and culture of ameloblast cells from human fetal tissues and co-culture of ameloblast cells and dental pulp cells. It is certainly a plus to have Drs. Richard Schneider and Renee Pera Reijo on board as collaborators/consultants. The proposal was well and logically described with realistic initial goals. WEAKNESSES: The reviewers had some concerns about feasibility given that there are many conceptual and technical problems in the proposal, reflecting PI’s inexperience in early tooth development. These concerns are detailed below. (1) The PI proposes to use Fgfr2, Pitx2 and Fgf8 as markers for epithelial lineage cells. These genes are indeed expressed in committed or differentiated dental epithelial cells, but are not dental epithelial specific markers. (2) Fgfr2 is known to be expressed in mouse dental epithelium. However, whether or not the human homolog exhibits a similar expression pattern is not known. The size of the proposed human Fgfr2 promoter is not indicated, and how the promoter activity and specificity will be determined is not provided. (3) The PI proposes to isolate oral epithelia from the first and second arches of E9 – 11.5 mouse embryos. Epithelial cells will be cultured, conditioned media collected and used as inducer for the induction of hESCs into dental epithelial lineage. It is well known that only the dental (and the presumptive dental) epithelium from E9.5 – 11.5 first arch or mandible possesses the odontogenic inductive capability. Epithelium from the SECOND arch never has such inductive capability. In addition, at E11.5, the second arch has already fused with the mandible. Indeed, previous studies demonstrate that the dental epithelia of E9 to E11.5 are able to induce tooth development. However, the signaling molecules released from the epithelium are believed to act on the adjacent mesenchyme instead of epithelial cells to initiate tooth developmental program. Lastly and most importantly, dental epithelial cells from early embryo, once cultured alone in the absence of mesenchymal cells, would loss their odontogenic fate quickly. (4) Growth factors known to be expressed in the dental epithelium, such as FGF4, Shh, Wnt4/10b, will be added into culture media to induce dental epithelial lineage cells from hESCc. It is not clear how these factors will be administrated. Will each of them be tested separately, or in a combination? (the ratio could be a critical issue). It should be pointed out that p63 is a transcription factor instead of a growth factor. It will never work when added in culture medium. (5) The PI proposes to induce hESCs to neural crest cells and then further into an odontogenic mesenchymal fate. Ncx-1 positive cells induced from hESCs will be harvested for odontogenic induction studies. It is known that only cranial neural crest cells can respond to the induction of dental epithelium to commit an odontogenic fate and develop a tooth. Ncx-1 is a molecular marker for neural crest and its derivatives, but it is not a specific one for cranial neural crest. (6) Pax9 expression will be assessed as differentiation of odontogenic mesenchymal cells. Again, Pax9 is not a specific marker for dental mesenchymal cells. The proposed human Pax9 promoter (size is not known) needs to be tested for its odontogenic expression specificity before it can be used. DISCUSSION: While this was an interesting idea, the reviewers had questions about feasibility and about whether cells could be differentiated to the desired state.