Small molecules direct the generation of ameloblast-like cells from human embryonic stem cells

Abstract

Abstract Background Ameloblasts present a promising avenue for the investigation of enamel and tooth regeneration. Previous protocols for directing the differentiation of human embryonic stem cells (hESCs) into dental epithelial (DE) cells involving the need for additional cells, conditional medium, and the use of costly cytokines. Importantly, ameloblasts have not been generated from hESCs in previous studies. Hence, we aimed to identify defined differentiation conditions that would solely utilize small molecules to achieve the production of ameloblasts. Methods We developed a three-step strategy entailing the progression of hESCs through non-neural ectoderm (NNE) and DE to generate functional ameloblasts in vitro. Initially, the NNE fate was induced from hESCs using a 6-day differentiation protocol with 1 µmol/L Retinoic acid (RA). Subsequently, the NNE lineage was differentiated into DE by employing a combination of 1 µmol/L LDN193189 (a BMP signaling inhibitor) and 1 µmol/L XAV939 (a WNT signaling inhibitor). In the final phase, 3 µmol/L CHIR99021 (a WNT signaling activator) and 2 µmol/L DAPT (a NOTCH signaling inhibitor) were utilized to achieve the fate of ameloblasts from DE cells. Three-dimensional cultures were investigated to enhance the ameloblast differentiation ability of the induced DE cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence were conducted to assess the expression of lineage-specific markers. Alizarin Red S (ARS) staining was performed to evaluate the formation of mineralization nodules. Results The application of RA facilitated the efficient generation of NNE within a six-day period. Subsequently, upon stimulation with LDN193189 and XAV939, a notable emergence of DE cells was observed on the eighth days. By the tenth day, ameloblast-like cells derived from hESCs were generated. Upon cultivation in spheroids, these cells exhibited elevated levels of ameloblast markers AMBN and AMELX expression, suggesting that spheroid culture augments the differentiation of ameloblasts. Conclusion We established an efficient small molecule-based method to differentiate hESCs into ameloblast-like cells through the concerted modulation of RA, BMP, WNT, and NOTCH signaling pathways, potentially advancing research in enamel and tooth regeneration.

Keywords

• Human embryonic stem cell
• Ameloblasts
• Small molecules
• Spheroids