O9-1 Mouse Cranial Neural Crest Cell Line

O9-1 Cells Contribute to Tooth Germ Formation in vitro

Damage or loss of teeth profoundly affects life quality, and restoring them conventionally relies on non-biological materials. NCCs, integral in embryonic development, have been explored for their potential in tissue regeneration. Zhang's team utilized O9-1 mouse cranial neural crest cell line and iPSC-derived neural crest-like cells (iNCLCs) to study their differentiation into odontoblasts and formation of dentin-pulp complexes.

To explore the role of various mesenchymal cell populations in forming bioengineered tooth germ, they mixed O9-1 cells, hDPSCs, or NIH3T3 with E14.5 dental mesenchymal cells at 25, 50, and 100% ratios with E14.5 dental epithelium (Fig. 1A). Tooth germ formation was observed in mixtures with 25% NIH3T3 and 25% or 50% O9-1 cells. No GFP-labeled NIH3T3 cells were found in the germ (Fig. 1B), indicating they don't contribute to formation. No tooth germ formed with hDPSCs at any ratio. To further examine O9-1 cells' role, they used red fluorescence-labeled dental epithelium. E14.5 dental germs were dissected, and mesenchyme was mixed with O9-1 cells at a 1:1 ratio (Fig. 2A and B). In organ cultures, red fluorescent epithelium and green GFP-labeled O9-1 cells were observed in the developing tooth germ (Fig. 2C and D). O9-1 cells integrated with the germ and showed characteristics of odontoblasts, confirmed by DMP-1 staining (Fig. 2D). This indicates that O9-1 cells, a mouse cranial neural crest cell line, can differentiate into odontoblasts and contribute to tooth germ formation.

Fig. 1. Schematicof tooth germ reconstitution and NIH3T3 cells failure to contribute to tooth germ formation (Zhang M, Zhang XC, et al., 2020).

Fig. 2. O9-1 cells contribute to tooth germ formation in vitro (Zhang M, Zhang XC, et al., 2020).

Overexpression of miR-129-5p and miR-340-5p Inhibits Cell Proliferation in MEPM and O9-1 Cells

Cleft lip and palate (CL/P) represent common birth defects that develop under the influence of genetic and epigenetic components combined with environmental elements. miRNAs are critical regulators of craniofacial development while also being linked to CL/P conditions. Yoshioka et al. explored whether suppressing specific miRNAs, particularly miR-124-3p and miR-340-5p, can prevent cleft palate induced by all-trans retinoic acid (atRA) in mice, offering a potential preventive strategy for CL/P.

To examine their role in orofacial clefts, they first treated primary MEPM cells, primary mouse embryonic lip mesenchymal (MELM) cells and O9-1 mouse cranial neural crest cells, an established mouse neural crest cell line, with either a miR-129-5p or miR-340-5p mimic. These mimics inhibited cell proliferation in palatal and O9-1 cells but not lip cells (Fig. 3A, B and Fig. 4A), indicating their role in palatal shelf growth. Next, to identify target genes regulated by miR-129-5p and miR-340-5p, we performed quantitative RT-PCR analysis for genes associated with CP in MEPM and O9-1 cells. miR-129-5p regulated Sox5, Tfap2a, Trp53, Zeb1, and others, while miR-340-5p regulated Chd7, Fign, and Tgfbr1 (Fig. 3C, D and Fig. 4B, C). Although miRNA inhibitors didn't affect proliferation in MEPM cells, they upregulated target genes, implying controlled gene regulation without promoting proliferation (Fig. 3E-G and Fig. 4D-F).

Fig. 3. Overexpression of miR-129-5p and miR-340-5p suppresses cell proliferation and gene expression in MEPM and MELM cells (Yoshioka H, Suzuki A, et al., 2022).

Fig. 4. Effect of miR-129-5p and miR-340-5p on cell proliferation and gene expression in O9-1 cells (Yoshioka H, Suzuki A, et al., 2022).