Single cell RNA sequencing reveals human tooth type identity and guides in vitro hiPSC derived odontoblast differentiation (iOB)

• Published in: Frontiers in Dental Medicine Vol. 4
• Main Authors: Hanson-Drury, Sesha, Patni, Anjali P., Lee, Deborah L., Alghadeer, Ammar, Zhao, Yan Ting, Ehnes, Devon Duron, Vo, Vivian N., Kim, Sydney Y., Jithendra, Druthi, Phal, Ashish, Edman, Natasha I., Schlichthaerle, Thomas, Baker, David, Young, Jessica E., Mathieu, Julie, Ruohola-Baker, Hannele
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media SA 20.07.2023; Frontiers Media S.A
• Subjects: cell signaling; de novo designed mini protein binders; Dentistry; enamel knot; human tooth; odontoblast; RK1-715; single cell RNA sequencing; cell signaling4; human6; single cell sequencing2; tooth development8; odontoblast1; de novo designed mini binders5; enamel knot3; regenerative dentistry7
• ISSN: 2673-4915; 2673-4915
• DOI: 10.3389/fdmed.2023.1209503

Over 90% of the U.S. adult population suffers from tooth structure loss due to caries. Most of the mineralized tooth structure is composed of dentin, a material produced and mineralized by ectomesenchyme derived cells known as odontoblasts. Clinicians, scientists, and the general public share the desire to regenerate this missing tooth structure. To bioengineer missing dentin, increased understanding of human tooth development is required. Here we interrogate at the single cell level the signaling interactions that guide human odontoblast and ameloblast development and which determine incisor or molar tooth germ type identity. During human odontoblast development, computational analysis predicts that early FGF and BMP activation followed by later HH signaling is crucial. Here we generate a differentiation protocol based on this sci-RNA-seq analysis to produce mature hiPSC derived odontoblasts in vitro (iOB). Further, we elucidate the critical role of FGF signaling in odontoblast maturation and its biomineralization capacity using the de novo designed FGFR1/2c isoform specific minibinder scaffolded as a C6 oligomer that acts as a pathway agonist. Using computational tools, we show on a molecular level how human molar development is delayed compared to incisors. We reveal that enamel knot development is guided by FGF and WNT in incisors and BMP and ROBO in the molars, and that incisor and molar ameloblast development is guided by FGF, EGF and BMP signaling, with tooth type specific intensity of signaling interactions. Dental ectomesenchyme derived cells are the primary source of signaling ligands responsible for both enamel knot and ameloblast development.