Craniofacial cartilage organoids from human embryonic stem cells via a neural crest cell intermediate

Abstract Severe birth defects or major injuries to the face require surgical reconstruction and rehabilitation. The ability to make bona fide craniofacial cartilage – cartilage of the head and face – from patient-derived induced pluripotent stem cells (iPSCs) to repair these birth defects and injuries has tremendous translational applications, but is not yet possible. The neural crest is the normal developmental pathway for craniofacial cartilage, however, the knowledge of cell signaling pathways that drive neural crest differentiation into craniofacial chondrocytes is limited. Here we describe a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cells (hESCs) and IPSCs through a neural crest stem cell (NCSC) intermediate. Histological staining of cartilage organoids revealed tissue architecture typical of hyaline cartilage. Organoids were composed of rounded aggregates of glassy, gray matrix that contained scattered small nuclei in lacunae. Mass spectrometry shows that the organoids express robust levels of cartilage markers including aggrecan, perlecan, proteoglycans, and many collagens. Organoids expressed markers indicative of neural crest lineage, as well as growth factors that are candidates for chondrocyte differentiation factors. The data suggest that chondrocyte differentiation is initiated by autocrine loops driven by a combination of secreted growth factors that bind to chondrocyte receptors. Craniofacial cartilage organoids were continuously cultured for one year, reaching up to one centimeter in diameter. The ability to grow craniofacial cartilage from NCSCs provides insights into the cell signaling mechanisms of differentiation into craniofacial cartilage, which lays the groundwork for understanding mechanistic origins of congenital craniofacial anomalies and repairing cartilaginous structures of the head and face.