Remission for Loss of Odontogenic Potential in a New Micromilieu In Vitro.

Yunfei Zheng,Duanqing Pei,Dandan Yang,Lingfei Jia,Shubin Chen,Pengfei Liu,Lihong Ge,Andrew Paul Hutchins,Shicheng Wei,Jinglei Cai

doi:10.1371/journal.pone.0152893

Abstract

During embryonic organogenesis, the odontogenic potential resides in dental mesenchyme from the bud stage until birth. Mouse dental mesenchymal cells (mDMCs) isolated from the inductive dental mesenchyme of developing molars are frequently used in the context of tooth development and regeneration. We wondered if and how the odontogenic potential could be retained when mDMCs were cultured in vitro. In the present study, we undertook to test the odontogenic potential of cultured mDMCs and attempted to maintain the potential during culturing. We found that cultured mDMCs could retain the odontogenic potential for 24 h with a ratio of 60% for tooth formation, but mDMCs were incapable of supporting tooth formation after more than 24 h in culture. This loss of odontogenic potential was accompanied by widespread transcriptomic alteration and, specifically, the downregulation of some dental mesenchyme-specific genes, such as Pax9, Msx1, and Pdgfrα. To prolong the odontogenic potential of mDMCs in vitro, we then cultured mDMCs in a serum-free medium with Knockout Serum Replacement (KSR) and growth factors (fibroblastic growth factor 2 and epidermal growth factor). In this new micromilieu, mDMCs could maintain the odontogenic potential for 48 h with tooth formation ratio of 50%. Moreover, mDMCs cultured in KSR-supplemented medium gave rise to tooth-like structures when recombined with non-dental second-arch epithelium. Among the supplements, KSR is essential for the survival and adhesion of mDMCs, and both Egf and Fgf2 induced the expression of certain dental mesenchyme-related genes. Taken together, our results demonstrated that the transcriptomic changes responded to the alteration of odontogenic potential in cultured mDMCs and a new micromilieu partly retained this potential in vitro, providing insight into the long-term maintenance of odontogenic potential in mDMCs.

Highlights

Vertebrate organs, including tooth, develop upon interactions typically between epithelial and mesenchymal tissues, with one tissue component producing inductive stimuli and another one responding to the induction [1,2]
Loss of odontogenic potential in mouse dental mesenchymal cells Among the isolated Mouse dental mesenchymal cells (mDMCs), some displayed a spindle-shaped, fibroblast-like morphology and others an elliptic morphology when adhering to the plates (Fig 2A)
When recombined with E14.5 dental epithelium, both freshly isolated mDMCs and molar mesenchyme tissues developed into teeth with well-differentiated odontoblasts after 3 weeks of subrenal culture (Fig 2C)

Summary

Introduction

Vertebrate organs, including tooth, develop upon interactions typically between epithelial and mesenchymal tissues, with one tissue component producing inductive stimuli and another one responding to the induction [1,2]. Odontogenic potential represents an instructive induction capability of a tissue to induce gene expression in an adjacent tissue and to initiate tooth development [3]. The odontogenic potential shifts from the epithelial compartment to dental mesenchyme at the early bud stage [4,5]. Dental mesenchymal cells isolated from prenatal or postnatal tooth germs participate in whole-tooth regeneration in mice, pigs, and rats [10,11,12]. Several available cell sources potentially could be employed to regenerate a whole tooth, including ecto-mesenchymal cells prepared from postnatal teeth, immortalized cell lines, and induced pluripotent stem cells

Methods

Results

Conclusion